Process for protein production in plants

ABSTRACT

This invention provides for the secretion of heterologous protein in plant systems. In particular, this invention provides for the production of heterologous proteins by malting of monocot plant seeds. The heterologous genes are expressed during germination of the seeds and isolated from a malt. Also disclosed are chimeric genes, vectors and methods relating to the present invention. Protein production by cell culture techniques is also described.

This application is a continuation of PCT/US94/13179 filed 15 Nov. 1994,herein incorporated by reference, and a continuation-in-part of U.S.Ser. No. 08/153,563, filed 16 Nov. 1993, now U.S. Pat. No. 5,693,506herein incorporated by reference.

FIELD OF THE INVENTION

The field of this invention is production of polypeptides in germinatingplant seeds employing the malting process. The invention also relates toplant expression vectors, chimeric genes and plant cell culture methods.

BACKGROUND OF THE INVENTION

The ability to clone and produce a wide range of proteins from diversesources became feasible with the advent of recombinant technology. Theselection of expression hosts for commercial biotechnology proteins isbased on the economics of fermentation and purification as well as theability of the host to accomplish the post-translational modificationsneeded for full biological activity of the recombinant protein. Some ofthese post-translational modifications include: signal peptideprocessing, pro-peptide processing, protein folding, disulfide bondformation, glycosylation, gamma carboxylation and beta-hydroxylation.Some of the economic factors influencing the choice of an expressionhost include: rates of biomass production, equipment costs, mediumcomposition and expense, processes for protein recovery andpurification, product yields, and the potential for contamination.

Much of the early work in biotechnology was directed toward theexpression of recombinant or "heterologous" proteins in prokaryotes,like Escherichia coli and Bacillus subtilis. Such work in procaryotesprovided ease of genetic manipulation, growth of the organisms in batchculture and the possibility of large-scale fermentation.

E. coli can perform signal peptide processing, protein folding, anddisulfide bond formation. However, it cannot secrete proteinsextracellularly glycosylate, gamma carboxylate, beta hydroxylate orprocess propeptides. B. subtilis suffers from the same limitations E.coli except that it is capable of extracellular secretion.

Total production costs from bacteria are also high because of problemswith product recovery, purification, and the inability of bacteria toperform many of the post-translational modifications mentioned above.Furthermore, E. coli and other bacteria are pathogens and contaminants,such as, pyrogens and endotoxins, must be removed from the recombinantlyproduced protein. In addition, extensive post-purification chemical andenzymatic treatments (e.g., to refold the protein into an active form)can be required to obtain biologically active protein.

Because proteins are not secreted from prokaryotes, like E. coli, suchcells must be disrupted for product recovery. The subsequent release ofbacterial contaminants and other proteins make product purification moredifficult and expensive. Because purification accounts for up to 90% ofthe total cost of producing recombinant proteins in bacteria, proteins,like tissue Plasminogen Activator (tPA), can cost several thousanddollars per gram to produce from E. coli.

Because of the many inadequacies associated with prokaryotic hosts, thebiotechnology industry has looked to eukaryotic hosts like mammaliancell tissue culture, yeast, fungi, insect cells, and transgenic animals,to properly and efficiently express recombinant proteins. However, thesehosts can suffer from any or all of the following disadvantages:expensive fermentation, low yields, secretion problems, inappropriatemodifications in protein processing, high operating costs, difficultiesin scaling up to large volumes, and/or contamination that either killsthe host culture or makes product purification more expensive. For thesereasons, existing eukaryotic hosts are unable to provide high-volume,low-cost protein production of recombinant proteins.

For most of those proteins requiring extensive post-translationalmodifications for therapeutic and/or functional activity, mammalian cellculture is the most common alternative to E. coli. Although mammaliancells are capable of correctly folding and glycosylating bioactiveproteins, the quality and extent of glycosylation can vary withdifferent culture conditions among the same host cells. Furthermore,mammalian culture has extremely high fermentation costs (60-80% of totalproduction expense), requires expensive media, and poses safety concernsfrom potential contamination by viruses and other pathogens. Yields aregenerally low, for example, in the range of 0.5-1.5% of cellularprotein, or up to about 300-400 milligrams per liter.

Yeast, fungi, insect cells and transgenic animals are currently beingused as alternatives to mammalian cell culture. Yeast, however, producesincorrectly glycosylated proteins that have excessive mannose residuesand generally limited eukaryotic processing. Further, although thebaculovirus insect cell system can produce high levels of glycosylatedproteins, these are not secreted--making purification complex andexpensive. Fungi represent the best current system for high-volume,low-cost production, but they are not capable of expressing many targetproteins. Transgenic animals are subject to lengthy lead times todevelop herds with stable genetics, high operating costs, andcontamination by animal viruses.

The biochemical, technical and economic limitations on existingprokaryotic and eukaryotic expression systems has created substantialinterest in developing new expression systems for recombinant proteins.Plants represent the most likely alternative to existing systems becauseof the advantageous economics of field-grown crops, the ability tosynthesize proteins in storage organs like tubers, seeds, fruits andleaves and the ability of plants to perform many of thepost-translational modifications previously described. However, existingplant expression systems suffer from low yield (<1.5% of total cellularprotein).

Furthermore, expression of the target protein occurs in the open field(in roots, stems, leaves, fruits and seeds), thus making it difficult toprevent the recombinant protein from entering the food and feed chain.This is an issue of much concern to government regulatory agencies.

Although the use of plant cell culture to express proteins has beendiscussed, the lack of knowledge about the genetics and biochemistry ofplant gene expression and secretion has precluded this system from beingdeveloped into a commercially feasible one.

RELEVANT LITERATURE

The potential for the use of plant cell cultures to product proteins hasbeen described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptionsof the rice amylase genes may be found in Huang, et al., Proc. Natl.Sci. U.S.A. 89:7526-7530 (1992); Huang, et al., Plant, Molecular Biology14:655-668 (1990); Huang, et al., Nucleic Acids Research 18:7007-7014(1990); Huang, et al., Gene 11 1:223-228 (1992); Rodriguez, et al.:Organization Structure and Expression of the Rice α-Amylase MultigeneFamily. Second International Rice Genetics Symposium. Rice Genetics11:417-429 (1990); Sutliff, et al., Plant Molecular Biology 16:579-591(1991). The promoter sequences for the rice amylase genes are describedin Huang, et al., Nucleic Acids Research 18:7007-7014 (1990).Descriptions of plant protein signal peptides may be found in additionto the references described above in Vaulcombe, et al., Mol. Gen. Genet.209:33-40 (1987); Chandler, et al., Plant Molecular Biology 3:407-418(1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein , et al.,Gene 55:353-356 (1987); Whittier, et al., Nucleic Acids Research 1 5:2515-2535 (1987); Wirsel, et al., Molecular Microbiology 3:3-14 (1989); Yu,et al., Gene 122:247-253 (1992).

A description of the regulation of plant gene expression by thephytohormone, gibberellic acid and secreted enzymes induced bygibberellic acid can be found in R. L. Jones and J. MacMillin,Gibberellins: in: Advanced Plant Physiology,. Malcolm B. Wilkins, ed.,1984 Pitman Publishing Limited, London, pp. 21-52. References thatdescribe other metabolically-regulated genes: Sheen, Plant Cell,2:1027-1038(1990); Maas, et al., EMBO J. 9:3447-3452 (1990); Benkel andHickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987).

SUMMARY OF THE INVENTION

The present invention has two aspects. In the first aspect, theinvention describes the production of high levels of recombinantproteins by seeds during the malting process. The invention includes amethod of producing a protein or polypeptide in a monocot plant seed.Monocot plant seeds typically contain an endosperm surrounded by analeurone or scutellar epithelium layer. Embryo development takes placewithin the endosperm. In the method, seeds are provided that contain achimeric gene having at least the following components: (i) atranscriptional regulatory region inducible during seed germination,(ii) a heterologous first DNA sequence encoding the protein, and (iii) asecond DNA sequence encoding signal polypeptide. The second DNA sequenceis operably linked to the transcriptional regulatory region and thefirst DNA sequence. The signal polypeptide encoded by the second DNAsequence is in translation-frame with the protein and is effective tofacilitate secretion of the protein across the aleurone or scutellarepithelium layer into the endosperm. The seeds are malted underconditions to induce expression of the transcriptional regulatory regionand production of the protein or polypeptide of interest.

In this method, the leader sequence may be omitted from the chimericgene construct if secretion of the protein of interest is not required,for example, for production of a mash or malted seed product intendedfor veterinary use.

In the above method, the seed may be obtained from a cereal plant,including, but not limited to wheat, rice, oats, rye, corn, sorghum,millet or barley. Barley and rice seeds are preferred embodiments.

The transcriptional regulatory region of the chimeric gene used in thepractice of the present invention can be obtained, for example, from oneof the following groups: α-amylase genes, sucrose synthase genes andsucrose-6-phosphate synthetase genes. Examples of α-amylase genesinclude the following genes obtained from rice and their homologs inother monocots: RAmy1A (SEQ ID NO:2), RAmy3B, RAmy3C, RAmy3D, HV18 (SEQID NO:1) and RAmy3E. In one embodiment of the invention the RAmy3D geneor homologs thereof is used.

Expression of the transcription regulatory region of the chimeric genecan be specifically regulatable by a small molecule. Examples of suchsmall molecules include plant hormones, cytokines and metabolites.Exemplary small molecules include, but are not limited to, absisic acid,gibberellic acids, indoleacetic acids, kinetins, butyric acid, oxalicacid, acetic acid, okadakic acid or arachidonic acid. In one embodiment,the transcription regulatory region is from the RAMY3D gene, or ahomolog thereof, and the small molecule is absisic acid.

The malting step of the present method can also include the addition orremoval of a small molecule in an amount effective to stimulateproduction of the protein.

Other embodiments of the transcriptional regulatory region of thechimeric gene includes regulatory regions sucrose synthase genes orsucrose-6-phosphate synthetase genes. With these promoters usefulregulatory small molecules include sugar (e.g., glucose or sucrose),sugar-phosphates, or other sugar-derivative molecules.

The protein produced by the method of the present invention can beselected from any number of sources. Exemplary classes of proteins orpolypeptides include, but are not limited to, the following: enzymes,antibodies, growth factors, cytokines, hormones, or antigens (e.g.,vaccines). Specific proteins or polypeptides include, but are notlimited to, the following: α-antitrypsin, antithrombin 3, fibrinogen,human serum albumin, factor VIII, granulocyte colony-stimulating factorand granulocyte macrophage colony-stimulating factor. The protein mayalso be an industrial protein (e.g., xylanase, oxidoreductase,peroxidase, glucanase, α-amylase, phytase or glucose oxidase).

The signal polypeptide (or signal sequence) can be obtained from anumber of sources. Exemplary gene-sources for the signal sequence areα-amylase genes, sucrose synthase genes and sucrose-6-phosphatesynthetase genes. Examples of α-amylase genes include the followinggenes obtained from rice and their homologs in other monocots: RAmy1A(SEQ ID NO:2), RAmy3B, RAmy3C, RAmy3D, HV18 (SEQ ID NO:1) and RAmy3E.

The method of the present invention can also include isolation orpartial purification of the protein or polypeptide of interest.

In another embodiment, the method of the invention includes separationof the embryo from the endosperm of the seed. In this embodiment, theembryo is (i) separated from the other seed parts, and (ii) germinatedunder conditions that induce expression of the transcriptionalregulatory region and production of the protein. The seeds used in themalting process may have one or more chimeric genes, where the geneshave the same or different transcription regulatory regions. The methodmay include separating the embryos and germinating them under conditionsthat (i) induce expression of the transcriptional regulatory region ofthe second chimeric gene, and (ii) production of the polypeptide of thesecond chimeric gene. The transcriptional regulatory region can, forexample, be selected from those described above. The method may alsoinclude separating embryos of the seed from the endosperm, contactingthe endosperm with the plant hormone which induces expression of theexpression cassette, and isolating the heterologous protein from theendosperm.

Also included in the present invention are the germinated seed, maltedseed and mash products by the above-described method. These products maybe directly used in veterinary applications where purification of theexpressed protein or polypeptide is not required (e.g., growth hormoneor vaccines).

In another aspect the invention includes the above-described chimericgene constructs. Further, the invention includes plant expressionvectors carrying the chimeric genes, plant cells bearing the chimericgene or transformed by such vectors, and transgenic plants bearing thechimeric gene. The transgenic plants and plant cells can, for example,be selected from the following group: rice, wheat, oats, rye, corn,sorghum, millet, and barley.

The present invention also includes transgenic seeds produced by thetransgenic plants of the present invention. Such transgenic seeds carrythe chimeric gene of the present invention.

In the second aspect of the present invention, a plant cell culturesystem is described for the production of recombinant proteins. Thecells for the culture system are preferably derived from scutellartissue. The cells used in this aspect of the present invention aretransgenic and contain a chimeric gene. The chimeric gene contains atleast the following elements: (i) a transcription regulatory regioninducible during seed germination, where expression mediated by theregion is specifically regulatable by a small molecule, and (ii) aheterologous DNA sequence that encodes the polypeptide, where the DNAsequence is operably linked to transcription regulatory region orpromoter.

The chimeric genes can be constructed using the components describedabove, including the transcription regulatory region, proteins orpolypeptides for expression, and regulatory small molecules.

The invention includes plant expression vectors carrying the chimericgenes, plant cells bearing the chimeric gene or transformed by suchvectors, and transgenic plants bearing the chimeric gene. The transgenicplants and plant cells can, for example, be selected from the followinggroup: rice, wheat, oats, rye, corn, sorghum, millet, and barley.

In one embodiment, the second aspect of the invention includes a methodfor modulating expression of a polypeptide in plant tissue cell culture.In this method the cells are the transgenic cells just described,typically from scutellar epithelium. These cells, carrying the chimericgene, are cultured under conditions that facilitate plant cell growth.Expression of the transcription regulatory region is modulated byaddition or removal of the regulatory small molecule to the plant cellculture.

Cells used in this aspect of the invention are monocot cells from, forexample, wheat, rice, oats, rye, corn, sorghum, millet or barley.

Modulation of expression can be accomplished by inhibition or induction.In one embodiment, the modulation is an inhibition of expression and thesmall molecule is a sugar or sugar-phosphate derivative. In anotheraspect, the modulation is an induction of expression and the smallmolecule is a plant hormone. Selected plant hormones can also be used toinhibit expression.

In the method, the culturing step can further include the addition orremoval of sugar or sugar-derivatives in an amount effective tostimulate production of the heterologous protein. In another embodiment,modulation is the enhancement of expression of the polypeptide in planttissue cell culture and the enhancement is accomplished by addition of asmall molecule to the plant cell culture, where the small moleculespecifically induces expression from the transcription regulatoryregion. An exemplary embodiment of this type of modulation is the use ofthe transcription regulatory region from the RAMY3D gene, or a homologthereof, and where the small molecule is absisic acid.

This aspect of the invention includes production and isolation orpartial purification of the protein or polypeptide of interest.

The invention also includes the following method of producing apolypeptide in plant cells. The transgenic plant cells described aboveare cultured in vitro (i) under conditions that facilitate plant cellgrowth, and (ii) in the presence of a small molecule that inhibitsexpression from the transcription regulatory region. The small moleculeis present at a concentration effective to repress expression from thetranscription regulatory region. The concentration of this smallmolecule is then reduced in the culture to a level that permitsexpression from the transcription regulatory region and allowsproduction of the polypeptide. Exemplary small molecules for thepractice of this embodiment of the invention include sugars orsugar-phosphate derivatives. After removal of the inhibitory smallmolecule, a second small molecule can be added to induce expression fromthe promoter present in the chimeric gene.

The invention also includes (i) an isolated DNA sequence essentiallyconsisting of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2;an isolated DNA sequence essentially consisting of a transcriptionalregulatory region obtained from SEQ ID NO:1 or SEQ ID NO:2; and (iii)one of these isolated DNA sequences operably linked to a heterologousDNA sequence encoding a protein. Such isolated DNA sequences may alsoinclude a second DNA sequence encoding signal polypeptide. The secondDNA sequence is operably linked to the transcriptional regulatory regionand the heterologous DNA sequence. Further, the encoded signalpolypeptide is in translation-frame with the protein encoded by theheterologous DNA sequence.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a full color photograph of the expression of the GUS gene incell culture. FIG. 1B is a full color photograph of the expression ofthe GUS gene in seed.

FIG. 2 is a depiction of the plasmid construction for producingtransgenic seed with a RAmy3D promoter.

FIGS. 3A and 3B show two Southern blot hybridizations, where FIG. 3Adepicts DNA isolated from control (lane 1) and 3DG cells (lanes 2-5) andprobed with a GUS gene (GUS) probe, and FIG. 3B represents the same DNAfractions probed with a rice α-amylase gene (AMY) probe.

FIG. 4 provides the results of a fluorescent assay detecting GUS.

FIG. 5 provides the results of a fluorescent assay detecting GUS fromcells free of sucrose.

FIGS. 6A to 6C provide a comparison of G+C content for three differentα-amylase promoters, and FIG. 6D is a table comparing sequence homologybetween α-amylase promoters.

FIG. 7A depicts the construct of two gene fusions used to express GUS inrice seed. FIGS. 7B and 7C show the results of Southern blotsdemonstrating stable transmission of the GUS gene to rice progeny.

FIGS. 8A and 8B are fluorometric measures of the expression of GUS usingthe H4 (FIG. 8A) and E4 (FIG. 8B) constructs in different transgenicrice seed.

FIGS. 9A to 9D are depictions of fluorometric measures of the expressionof GUS in two H4/GUS transgenic lines (T21 and N33) (FIGS. 9A and 9B),and two E4/GUS transgenic lines (K43 and T62) (FIGS. 9C and 9D).

FIGS. 10A-10D provide graphic depictions of four fluorometric measuresof the expression of GUS in two H4/GUS transgenic lines (T21 and N33)(FIGS. 10A and 10B), and two E4/GUS transgenic lines (K43 and T62)(FIGS. 10C and 10D), quantifying the induction of GUS by addition ofgibberellic acid.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

"Cell culture" refers to cells and cell clusters both protoplast andcallus tissue that are without differentiated cells or organs and aregrowing on a growth media.

"In an amount effective" refers to an amount that is suitable to producethe desired effect in a measurable and reproducible amount.

"Inducible" means a promoter that is turned on by the presence orabsence of a cell hormone or metabolite. It includes both indirect anddirect inducement.

"Inducible during germination" refers to promoters which aresubstantially silent but not totally silent prior to germination but areturned on substantially (greater than 25%) during germination anddevelopment in the seed. Examples of promoters that are inducible duringgermination are presented below.

"Homologous DNA" refers to DNA not introduced into the plant or planthost by recombinant means.

"Heterologous DNA" refers to DNA which has been transfected into plantcells. Typically, heterologous DNA refers to DNA that is not originallyderived from the transfected or transformed cells' genomic DNA (e.g.,GUS gene sequences). A DNA sequence may also be heterologous to anadjacent DNA sequence, for example, a DNA sequence is heterologous to apromoter or transcription regulatory region, when the promoter ortranscription regulatory region is not native, that is, not naturallyoccurring adjacent the DNA sequence of interest.

A "transcription regulatory region" typically refers to nucleic acidsequences that influence and/or promote initiation of transcription,such as promoters. Promoters are typically considered to includeregulatory regions, such as enhancer or inducer elements.

A "chimeric gene," in the context of the present invention, typicallycomprises a promoter sequence operably linked to non-homologous DNAsequences that encode a gene product (e.g., a RAmy3D promoter adjacentDNA sequences encoding human serum albumin). A chimeric gene may alsocontain further transcription regulatory elements, such as transcriptiontermination signals, as well as translation regulatory signals, such as,termination codons.

"Operably linked" refers to components of a chimeric gene or anexpression cassette that function as a unit to express a heterologousprotein. For example, a promoter operably linked to a heterologous DNA,which encodes a protein, promotes the production of functional mRNAcorresponding to the heterologous DNA.

A "product" encoded by a DNA molecule includes, for example, RNAmolecules and polypeptides.

Two nucleotide sequences are considered to be "functionally homologous"if they hybridize with one another under moderately stringentconditions, i.e. 0.1% SSC at room temperature. Typically, two homologousnucleotide sequences are greater than or equal to about 60% identicalwhen optimally aligned using the ALIGN program (Dayhoff, M. O., in ATLASOF PROTEIN SEQUENCE AND STRUCTURE (1972) Vol. 5, National BiomedicalResearch Foundation, pp. 101-110, and Supplement 2 to this volume, pp.1-10.).

Two amino acid sequences are considered "homologous" if their aminoacids are greater than or equal to about 60% identical when optimallyaligned using the ALIGN program mentioned above.

"Removal" in the context of a metabolite includes both physical removalas by washing and the depletion of the metabolite through the absorptionand metabolizing of the metabolite by the cells.

"Signal sequence suitable to permit the heterologous protein to besecreted across the aleurone or scutellular epithelium" refers to anynaturally occurring signal sequence in monocots, dicots, animals ormicroorganisms that can permit a protein to be secreted from the cellsacross the stated organs of the monocot seed. Generally, a signalsequence is a sequence of amino acids that promotes the secretion of aprotein from a cell.

"Small molecules" are typically less than about 1 kilodalton and arebiological, organic, or even inorganic compounds (i.e., cisplatin).Examples of such small molecules include sugars, sugar-derivatives(including phosphate derivatives), and plant hormones (such as,gibberellic or absisic acid).

"Specifically regulatable" refers to the ability of a small molecule topreferentially affect transcription from one promoter or group ofpromoters (e.g., the α-amylase gene family), as opposed to non-specificeffects, such as, enhancement or reduction of global transcriptionwithin a cell by a small molecule.

"Substantially isolated" is used in several contexts and typicallyrefers to the at least partial purification of a protein or polypeptideaway from unrelated or contaminating components (e.g., plant proteinsremoved from a sample containing human α-1-antitrypsin). Methods andprocedures for the isolation or purification of proteins or polypeptidesare known in the art.

II. Production of Recombinant Proteins Using the Malting Process.

The invention described herein has essentially two distinct aspects. Inthe first aspect, the present invention relates to the discovery of theregulated expression of recombinant proteins in malted cereal seeds.Malting is the process by which grain, typically barley or rice, isgerminated under controlled conditions and in contained facilities toproduce a product that can be used for human consumption, animal feedand the brewing of alcoholic beverages. The process begins by steepingbarley seeds in 55° F. water for 48 hours followed by a four-daygermination of the grain in malting bins or drums.

During this time, the starchy portion of the seed, or endosperm, isconverted to maltose and other sugars. Maltsters use water, air and, insome instances, phytohormones, like gibberellic acid, to controltemperature and optimize the malting process. The malted grain is thenkiln-dried at temperatures between 120° F. and 130° F. to terminategermination and remove moisture. At this point the malted grain can bestored or sold to the food, feed or brewing industries. Products of themalting process include mashes and formulated seed products. During theprocess of malting germinated seeds are also produced.

In the malting process, the rapid conversion of starch to sugar isaccomplished by a tremendous burst of gene activity that results in theexpression of a starch degrading enzyme called α-amylase. During thepeak stage of germination, α-amylase is the major protein in the seed,constituting up to 60% of the total protein of the cells that surroundthe starchy endosperm. Alpha-amylase is secreted out of these cells andinto the endosperm where it digests the starch into sugar.

Expression and secretion of α-amylase during germination is so abundantthat it can be purified and sold as a research reagent for approximately$0.10 per gram. The compositions and methods described herein allowlow-cost, high-volume eukaryotic production of selected gene products,such as, proteins and polypeptides, based on the malting of transgenicseeds.

This aspect of the invention includes a method of producing a protein ina monocot plant seed. Monocot plant seeds contain an endospermsurrounded by an aleurone and scutellar epithelium layer. The embryotypically begins germination and development within the endosperm. Seedsare produced, for example, from transgenic plants, that contain achimeric gene having at least the following components:

(i) a transcriptional regulatory region inducible during seedgermination, several such regions are described in detail below,including promoters from α-amylase, sucrose synthase, andsucrose-6-phosphate synthetase genes;

(ii) a heterologous first DNA sequence encoding a gene product ofinterest, for example, a polypeptide or protein. Exemplary DNA codingsequences are described below; and

(iii) a second DNA sequence encoding signal sequence where the secondDNA sequence is operably linked to the transcriptional regulatory regionand the first DNA sequence. Further, the signal polypeptide sequence is(a) in translation-frame with the protein or polypeptide of interest and(b) effective to facilitate secretion of the protein or polypeptideacross said aleurone or scutellar epithelium layer into the endosperm.

These seeds are malted under conditions to induce expression of thetranscriptional regulatory region and production of the protein orpolypeptide of interest.

The plants (including organs, seeds, tissues and cells) used in theprocess of the present invention are derived from monocots, particularlythe members of the taxonomic family known as the Gramineae. This familyincludes all members of the grass family of which the edible varietiesare known as cereals. The cereals include a wide variety of species suchas wheat (Triticum sps.), rice (Oryza, sps.) barley (Hordeum sps.) oats,(Avena sps.) rye (Secale sps.), corn (Zea sps.) and millet (Pennisettumsps.).

Plant cells or tissues derived from the members of the Gramineae and aretransformed with expression constructs (i.e., plasmid DNA into which thegene of interest has been inserted) using a variety of standardtechniques (e.g., electroporation, protoplast fusion or microparticlebombardment). In one embodiment, the expression construct includes atranscription regulatory region (promoter) whose transcription isspecifically regulated by the presence or absence of a small molecule.

In one embodiment, the gene encoding the recombinant protein is placedunder the control of a metabolically regulated promoter. Metabolicallyregulated promoters are those in which mRNA synthesis or transcription,is repressed or induced by sugars or sugar derivatives. Examples ofmetabolically regulated promoters include those that transcribe some ofthe cereal α-amylase genes and sucrose synthase genes.

Another expression construct uses a hormonally regulated promoter toachieve expression of the recombinant protein in the germinated ormalted seed. Hormonally regulated promoters are those in which mRNAsynthesis or transcription, is repressed or induced by small molecules,like phytohormones such as gibberellic acid or absisic acid. Other suchsmall molecules include, but are not limited to, indoleacetic acids,kinetins, butyric acid, oxalic acid, acetic acid, okadakic acid andarachidonic acid.

Examples of hormonally regulated promoters include those that transcribesome of the cereal α-amylase genes. The promoters relevant to thisapplication include, but are not limited to the following: thosecontrolling the expression of the rice (Oryza sativa) α-amylases genes,RAmy1A, RAmy3D and RAmy3E; the barley α-amylase gene promoter, HV18; andthe sucrose synthase and sucrose-6-phosphate-synthetase (SPS) promotersfrom rice and barley.

Expression constructs utilize additional regulatory DNA sequences (i.e.,signal peptide sequences and preferred translational start codons) topromote efficient translation and extracellular secretion of the targetprotein. By fusing the genes for recombinant proteins to this array ofregulatory DNA sequences, the expression of recombinant proteins ingerminated transgenic seeds is placed under the transcription andsecretion control of a metabolically regulated or hormonally regulatedpromoter.

Cells or tissues or derived from cereal plants can be transformed singlyor together (i.e., co-transformation) with the expression constructs.From such transformed cells transgenic plants can be regenerated. Thesetransgenic plants are grown, allowed to produce seeds and therecombinant protein encoded by the expression construct can be recoveredfrom malted transgenic seeds.

The principle of using different cereal α-amylase promoters to express arecombinant protein in transgenic seeds is illustrated in FIG. 1B. Inthis figure, the gibberellic acid-induced promoter for the RAmy1A gene,was used to express the bacterial reporter gene, gusA, in rice (Example2). The gusA gene encodes the enzyme, beta-glucuronidase (GUS), thatproduces a blue chromophore in tissues expressing the gene. Thischromophore can be easily detected using a histochemical stainingmethod. In transgenic rice seeds containing the RAmy1A promoter/GUSfusion, the blue chromophore increases up to six days of germination.

Using the chimeric genes, vectors and methods of the present invention,cereal species such as rice, corn, wheat, oats, rye, barley and variousgrasses can be genetically engineered to express a wide range ofrecombinant proteins. By combining the technology of the presentinvention with well-established production methods (e.g., cropcultivation, malting, and product recovery), recombinant protein can beefficiently and economically produced for the biopharmaceutical,industrial processing, animal health and bioremediation industries.

The fact that this expression system does not require the use of geneticelements derived from animal or plant pathogens should facilitateregulatory acceptance.

A. Plant Expression Vectors

Expression vectors for use in the present invention comprise a chimericgene (or expression cassette), designed for operation in plants, withcompanion sequences upstream and downstream from the expressioncassette. The companion sequences will be of plasmid or viral origin andprovide necessary characteristics to the vector to permit the vectors tomove DNA from bacteria to the desired plant host.

The basic bacterial/plant vector construct will preferably provide abroad host range prokaryote replication origin; a prokaryote selectablemarker; and, for Agrobacterium transformations, T DNA sequences forAgrobacterium-mediated transfer to plant chromosomes. Where theheterologous gene is not readily amenable to detection, the constructwill preferably also have a selectable marker gene suitable fordetermining if a plant cell has been transformed. A general review ofsuitable markers for the members of the grass family is found in Wilminkand Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologoussequence into the plant genome are also recommended. These might includetransposon sequences, and the like, for homologous recombination, aswell as Ti sequences which permit random insertion of a heterologousexpression cassette into a plant genome.

Suitable prokaryote selectable markers include resistance towardantibiotics such as ampicillin or tetracycline. Other DNA sequencesencoding additional functions may also be present in the vector, as isknown in the art.

The constructs of the subject invention will include the expressioncassette for expression of the protein(s) of interest. Usually, therewill be only one expression cassette, although two or more are feasible.The recombinant expression cassette contains, in addition to theheterologous protein encoding sequence, at least the following elements:a promoter region, plant 5' untranslated sequences, initiation codondepending upon whether or not the structural gene comes equipped withone, and transcription and translation termination sequences. Uniquerestriction enzyme sites at the 5' and 3' ends of the cassette allow foreasy insertion into a pre-existing vector. These elements are discussedin detail below.

1. Heterologous Coding Sequences.

The heterologous coding sequence may be for any protein of interest,either prokaryotic or eukaryotic, particularly eukaryotic. The geneproviding the desired product will particularly be those genesassociated with large volume products. Therefore, products of particularinterest include but are not limited to enzymes, such as chymosin,proteases, polymerases, saccharidases, dehydrogenases, nucleases,glucanase, glucose oxidase, α-amylase, oxidoreductases (such as fungalperoxidases and laccases), xylanases, phytase, cellulase, hemicellulase,and lipase. More specifically, the invention can be used to produceenzymes such as those used in detergents, rennin, horse radishperoxidase, amylases from other plants, soil remediation enzymes, andother such industrial proteins.

Other proteins of interest are mammalian proteins. Such proteinsinclude, but are not limited to blood proteins (such as, serum albumin,Factor VII, Factor VIII (or modified Factor VIII), Factor IX, Factor X,tissue plasminogen factor, Protein C, von Willebrand factor,antithrombin III, and erythropoietin), colony stimulating factors (suchas, granulocyte colony-stimulating factor (G-CSF), macrophagecolony-stimulating factor (M-CSF), and granulocyte macrophagecolony-stimulating factor (GM-CSF)), cytokines (such as, interleukins),integrins, addressins, selectins, homing receptors, surface membraneproteins (such as, surface membrane protein receptors), T cell receptorunits, immunoglobulins, soluble major histocompatibility complexantigens, structural proteins (such as, collagen, fibroin, elastin,tubulin, actin, and myosin), growth factor receptors, growth factors,growth hormone, cell cycle proteins, vaccines, fibrinogen, thrombin,cytokines, hyaluronic acid and antibodies.

While for the most part, the product will be a peptidic product, genesmay be introduced which may serve to modify non-peptidic productsproduced by the cells. These proteins, fragments thereof, usually of atleast about 30 amino acids, fused combinations, mutants, and syntheticproteins, whether the proteins may be synthetic in whole or in part, sofar as their sequence in relation to a natural protein, may be produced.

The present invention also provides the advantage that polypeptideuseful for veterinary use, such as, vaccines and growth hormones, may beproduced by the malting process of the present invention. The productsof the malting reaction, containing the polypeptide of interest, canthen be formulated into mash product or formulated seed product directlyuseful in veterinary applications.

2. Signal Sequences

Also included in chimeric genes used in the practice of the method ofthe present invention are signal secretion sequences. In addition toencoding the protein of interest, the chimeric gene also encodes asignal peptide that allows processing and translocation of the protein,as appropriate. The chimeric gene typically lacks any sequence thatmight result in the binding of the desired protein to a membrane.

Typically, the transciptional regulatory region (i.e., the transcriptioninitiating region) is derived from a gene whose product is expressed andtranslocated during germination. By employing the signal peptidehomologous to such a transcriptional regulatory region, translocation ofthe protein of interest is achieved. In this way, the protein(s) ofinterest are translocated from the cells in which they are expressed andmay be efficiently harvested.

While it is not required that the protein be secreted from the cells inwhich the protein is produced, this facilitates the isolation andpurification of the recombinant protein. Table 1 provides a list ofknown signal sequences from wheat, barley and rice. Typically, thesesignal sequences facilitate secretion of proteins expressed in seedsacross the aleurone or scutellar epithelium layer into the endosperm ofthe seed.

                  TABLE 1    ______________________________________    Signal peptides of α-amylase genes    B-, barley α-amylase genes. W-, wheat α-amylase genes;    #, signal peptide cleavage site determined by protein sequencing;    *, predicted signal peptide cleavage site; /, intron splice site;    ".", space inserted to maximize the alignment.    Genes        References    ______________________________________    RAmy1A       Plant Molecular Biology. 14:655-668                 (1990).    RAmy1B       Nucleic Acids Research. 18:7007-7014                 (1990).    aAmy10-c     Gene. 122:247-253 (1992).    W-Amyl/13 Mol.                 Gen. Genet. 209:33-40 (1987).    W-2128       Gene. 55:353-356 (1987).    B-pM/C       Plant Molecular Biology. 3:407-418                 (1984) and Journal of Biological                 Chemistry. 260:3731-3738 (1985).    B-gKAmy141   Plant Molecular Biology. 9:3-17                 (1987).    RAmy2A       Gene. 111:223-228 (1992).    W-Amy2/54    Mol. Gen. Genet. 209:33-40 (1987).    B-clone E    Plant Molecular Biology. 3:407-418                 (1984) and J. of Biological Chemistry                 260:3731-3738 (1985).    B-gKAmy155   Plant Molecular Biology. 3:407-418                 (1984) and Plant Molecular Biology.                 9:3-17 (1987).    B-Amy32b     Plant Molecular Biology. 3:407-418                 (1984) and Nucleic Acids Research                 15:2515-2535 (1987).    RAmy3A       Plant Molecular Biology. 16:579-591                 (1991).    RAmy3B       Plant Molecular Biology. 16:579-591                 (1991).    RAmy3C       Plant Molecular Biology. 16:579-591                 (1991).    RAmy3D       Nucleic Acids Research. 18:7007-7014                 (1990).    RAmy3E       Nucleic Acids Research. 18:7007-7014                 (1990).    W-Amy3/33    Mol. Gen. Genet. 209:33-40 (1987).    Taka-amylase Molecular Microbiology. 3:3-14 (1989).    ______________________________________

Those of skill can routinely identify new signal peptides. Plant signalpeptides typically have a tripartite structure, with positively-chargedamino acids at the N-terminal end, followed by a hydrophobic region andthen the cleavage site within a region of reduced hydrophobicity. Theconservation of this mechanism is demonstrated by the fact that cerealα-amylase signal peptides are recognized and cleaved in foreign hostssuch as E. coli and S. cerevisiae.

The flexibility of this mechanism is reflected in the wide range ofpolypeptide sequences that can serve as signal peptides. Thus, theability of a sequence to function as a signal peptide may not be evidentfrom casual inspection of the amino acid sequence. Methods designed topredict signal peptide cleavage sites identify the correct site for onlyabout 75% of the sequences analyzed. (See Heijne Gv: Cleavage-sitemotifs in protein targeting sequences. In: J. K. Setlow (eds) GeneticEngineering, Vol. 14. Plenum Press, New York (1992)).

Although, sequence homology is not always present in the signalpeptides, hydrophilicity plots demonstrate that the signal peptides ofthese genes are relatively hydrophobic.

3. Promoters

(a) Exemplary Transcription Regulatory Regions

The preferred transcription regulatory or promoter region is chosen soas to be relatively silent, except during seed germination. For example,the expression level in the seed cells is at least about 20 times theexpression level in other plant tissue during the growth of the plant.This type of transcriptional regulation can be achieved in various ways,including the following: (i) by using the 5'-non-coding regionassociated with a protein which is produced solely or substantiallysolely during seed germination, or (ii) by using the regulatory portionof such a transcriptional initiation region in conjunction with adifferent RNA polymerase binding region.

In referring to a "substantial absence of expression" at times otherthan seed germination, it is intended that expression be very low ornon-existent except during seed germination. That is, the expression ofthe protein of interest, encoded by the chimeric gene, is low so as (i)to not affect the growth of the plant or expend significant plantresources, (ii) to not diminish the vigor of the plant growth, and (iii)to allow for the plant and mash to be used for its intended purpose,depending on the protein of interest.

A number of proteins are normally secreted across either the aleurone orscutellum during seed germination and seed elongation. Some examples ofsecreted plant enzymes induced by gibberellic acid include, but are notlimited to the following: α-amylase, protease, ribonuclease,β-glucanase, esterase, acid phosphatases (such as p-nitrophenyl,phosphatase, ATPase, phytase, naphthol AS-B1 phosphatase, and GTPase),pentosanase, endoxylanase, xylopyranosidase, arabinofuranosidase,glucosidase, and peroxidase.

In view of the teachings of the present specification, one of skill inthe art can recognize and implement useful promoter/signal sequencecombinations for the practice of the present invention. Because many ofthe useful sequences are evolutionarily related, the conserved sequencesfacilitate the identification of new promoters and signal sequencesuseful in the practice of this invention.

Standard nucleic acid hybridization technology can be used to probelibraries of other monocots, using previously identified promoters andsignal sequences, to identify "homologs" in these monocots. For example,a gene having a promoter region of interest is selected, a probespecifically hybridizable to the gene is chosen, and traditionalcross-hybridization experiments are performed under varying solutionstringencies. This method has been employed using the rice RAmy1A as aprobe to identify a homolog promoter in barley; the barley promoter forHV18 (SEQ ID NO:1).

Polymerase chain reaction technology (PCR; Mullis, K. B., U.S. Pat. No.4,683,202, issued 28 Jul. 1987; Mullis, K. B., et al., U.S. Pat. No.4,683,195, issued 28 Jul. 1987) can also be used to identify homologs ofknown promoters, for example, amplify unknown promoter using PCR primersable to bind to conserved regions of a selected promoter or signalsequence. Examples of conserved sequences in the rice amylase promoterregions are provided in Table 2. The sequences for the rice promoterswere reported in Huang N., et al., 1990, Nucl. Acids Res. 18:7007-7014(1990) and the taka promoter from Aspergillus oryzae was reported inWirsel, 1989, Molecular Microbiology, 3:3-14.

                                      TABLE 2    __________________________________________________________________________    Conserved sequences in the RAmy3D, RAmy3E and Take-amylase    __________________________________________________________________________    promoters    31 bp RAmy3D            GAGACCGGGCCCCGACGCGGCCGACGCGGCG                                   SEQ ID NO: 3            ++++++++++++++++++++++    31 bp RAmy3E            GAGAGCTCGCGCCGCCTCGATCGGCGCGGCG                                   SEQ ID NO: 4    11 bp RAmy3D            TTCCGGCTTGC            SEQ ID NO: 5            ++++++++++    11 bp RAmy3E            TTGCGGCTTGC            SEQ ID NO: 6    Taka-amylase            CGGCCCGTCGGC           SEQ ID NO: 7    __________________________________________________________________________     The "+" symbols indicate positions at which the RAmy3D and RAmy3E     sequences are identical.

The situation in rice is demonstrative. In rice, the α-amylase isozymesare encoded by a family of nine genes (Table 3). They are referred to asRAmy1A, 1B, 1C, 2A, 3A, 3B, 3C, 3D and 3E. The Rice α-Amylase genes areclassified into three subfamilies (RAmy1, RAmy2, and RAmy3) (See Huang,et al., 1992, Proc. Natl. Acad. Sci. USA. 89:7526-7530 and Huang N, etal., 1990, Plant Molecular Biology, 14:655-668) based on DNA sequencesimilarities to α-amylase gene subfamilies in other cereal species.Eight members of the α-amylase gene family in rice have been isolatedand characterized. A partial cDNA sequence, presumably corresponding toRAmy1C, has been reported in Yu S-M, et al., 1992, Gene. 122:247-253.The α-amylase genes have been mapped to five different chromosomes inrice.

                  TABLE 3    ______________________________________    Alpha-amylase gene expression in rice tissues    Gene   Germinated              Developing                                           Cultured    Names  Seedlings  Root   Leaf  Seeds   Cells    ______________________________________    All Genes            100.sup.a 1      3     4       65    RAmy1A .sup. ++++.sup.b                      ++++   ++++  +++     ++    RAmy1B -          -      -     -       +    RAmy1C  -.sup.c   .sup. NA.sup.d                             NA    NA      NA    RAmy2A +          +      +     +       +    RAmy3A +          -      -     -       ++    Ramy3B ++         NA     NA    NA      NA    RAmy3C ++         NA     NA    NA      NA    RAmy3D ++         ++     ++    -       ++++    RAmy3E +++        +++    +++   ++++    +++    ______________________________________     .sup.a Relative mRNA levels for all amylase genes are normalized to the     level of expression observed in germinated seedlings by Northern blot     hybridization.     .sup.b Relative levels of mRNA for each gene as estimated from Northern     blot hybridization or RNAPCR experiments. The amount of PCR product is     indicated from the highest (++++) to the lowest (+). Minus signs (-)     indicate that no product was observed in the RNAPCR reaction.     .sup.c Lack of expression based on restriction digest of RNAPCR products.     .sup.d NA = Not Available

Embodiments of the present invention include promoter/signal sequencecombinations of RAmy 1A, 3D and 3E, where expression is at a high levelduring germination. The 5'-non-coding region of 3E is characterized by aregion conserved with 3D, which is a GC-rich sequence of 31 bases andcontains two CGGC repeats. There is also an 11 base sequence which isconserved which contains a single copy of the CGGC sequence.

The 3D and 3E α-amylase genes are subject to suppression of expressionby sugars, particularly sucrose, glucose, fructose, and maltose. Thus,during cell fermentation, premature expression of the desired productcan be avoided by employing a sugar, particularly, sucrose, in thegrowth medium. Thus, sucrose may be used as a carbon source by the cellsand, when the sucrose is exhausted, expression of the desired proteinswill be initiated.

Other transcription initiation regulatory regions that may be employedinclude those that are induced by sugar such as sucrose synthase.

Complex transcriptional initiation regions can be employed by using theregulatory portion of one transcriptional initiation region with the RNApolymerase binding region of a different gene. In this way, expressioncan be regulated while providing for a high level of expression.

A preferred embodiment of the present invention uses a promoter that isregulated during germination. For example, the hormones absisics acid(ABA) and gibberellic acid (GA) play important regulatory roles incontrol of α-amylase gene expression in cereal seeds. ABA, which issynthesized during grain filling, acts as a negative regulator oftranscription for α-amylase and many other genes. ABA levels drop in themature desiccated grain, thus relieving the inhibition of α-amylase andother genes required for germination.

Obviously up-regulation is desired for over expression of heterologousproteins and GA mediated promoters are a desired embodiment. Theprevailing model for GA regulation during cereal seedling developmentinvolves the diffusion of GA from the embryo to the aleurone layer. GAthen induces the synthesis of hydrolytic enzymes such as α-amylase. Inrice, GA stimulates α-amylase gene expression in aleurone tissues.

But not all α-amylase promoters are induced by GA and not all areinducible when present in undifferentiated cells, such as those used inculture or when removed from the intact seed. For example RAmy3D andRAmy3E in rice callus and cultured cells are unaffected by GA. Nosignificant change in levels of RAmy3D and RAmy3E expression wasdetected in rice callus treated with GA and there are reports thatcallus treated with paclobutrazol, an inhibitor of gibberellinbiosynthesis, produced the same amount of α-amylase protein as untreatedcallus.

Finally, callus cultures derived from seeds of the GA-deficient dwarfmutant, cv. Tan-ginbozu, produced the same levels of α-amylase geneexpression with or without exogenous GA treatment. This suggests thatRamy3D and Ramy3E gene expression in the scutellum and in cultured cellsis independent of GA regulation.

The GA independent promoters appear to be missing a short sequence thatis present in GA inducible promoters. DNA sequence comparisons haveidentified four short, conserved sequences in the cereal α-amylasepromoters. The TATA Box (CTATAAATAG) is the binding site required forRNA polymerase II to initiate transcription. The Pyrimidine Box(YCTTTTY) and Box I (TATCCAT) may be involved in the developmentalregulation of the genes in the scutellum and aleurone.

The GARE Box (GA-Responsive Element) (TAACRRA) is required forGA-induction and ABA-repression of α-amylase gene expression. The GAREBox (GA-Responsive Element) in the RAmy1A gene (genomic clone lOSg2) islocated at base-143 relative to the transcription start site. Expressionof the RAmy1A gene (cDNA clone pOS103) is stimulated 50-100 fold byexogenous GA. The absence of GARE Box sequences in the promoters of therice RAmy3D and RAmy3E genes is consistent with the GA-independentexpression of these genes as discussed above.

Alternatively the agent inducing expression can be a metabolite which iseither a sugar or phosphorylated sugar. For example, RAmy3D geneexpression is metabolically regulated in rice embryo tissues. Evidencefor this is based on studies in which seeds were moistened to initiateseedling development and harvested after 0 to 48 hours of incubation.Embryos dissected out of these seeds had low levels of expression forRAmy3D. This pattern of expression was reversed if embryos were firstremoved from the seed at time zero and incubated in water for 0 to 48hours. Under these conditions, RAmy3D expression increased to five timesthe level observed in the intact rice seed.

Addition of sugar to the incubation medium used for the isolated embryosrestored normal expression of the RAmy3D gene (see examples). A numberof sugars, including sucrose, glucose, fructose and maltose, were ableto repress RAmy3D gene expression in isolated embryos (Karrer andRodriguez, 1992, The Plant Journal, 2:517-523).

In rice cell suspension cultures, α-amylase enzyme activity increasesafter the depletion of sucrose from the medium. This increase isconsistent with the pattern of α-amylase mRNA accumulation, which alsoincreases dramatically after the culture medium is depleted of sugar.Cells transferred to sugar-free medium begin to produce elevated levelsof total α-amylase mRNA within four hours. (See Yu S-M, et al., 1991, JBiol. Chem. 266:21131-21137 (1991).

Dot blot hybridization and gene-specific probes have been used todemonstrate that sugar controls the expression of both RAmy3D and RAmy3Ein cultured cells. Cells were subcultured into medium with 1%, 3%, 6% or12% sucrose. RNA isolated from cells cultured for five days showed thatRAmy3D and RAmy3E expression was repressed at the higher sugarconcentrations. Expression of both of these genes was induced in thecells cultured in 1% sucrose, presumably after sugar was depleted fromthe medium by cell growth. Expression of the RAmy1A and RAmy3A genes wasunaffected by these treatments. Thus, expression of the RAmy3D andRAmy3E genes is metabolically regulated by the concentration of sugar inthe culture medium.

To confirm that this regulation is acting at the level of transcription,the RAmy3D promoter was linked to the GUS reporter gene. The amount ofGUS enzyme activity produced by the cells provides a convenient measureof the expression of the engineered RAmy3D promoter. The RAmy3Dpromoter/GUS gene construct was co-electroporated into protoplasts withanother plasmid containing the gene for hygromycin-resistance. Southernblot hybridization showed that hygromycin-resistant cell lines containedthe RAmy3D/GUS gene construct. GUS activity was reduced in transformedcell lines grown at elevated sucrose concentrations (see examples). GUSactivity increased starting eight hours after the cells were transferredto sugar-free medium. These data demonstrate that the GUS reporter geneis being regulated by the RAmy3D promoter in cell culture just as theendogenous RAmy3D gene is regulated in the rice seed.

The promoters of the RAmy3D and RAmy3E genes have little sequencesimilarity, but there are two conserved sequences in their promotersthat may be involved in the metabolic regulation of these genes. AGC-rich sequence of 31 bases in RAmy3D (Table 2) contains two CGGCrepeats (underlined). This tandem repeat structure within the 31 basesequence is similar to the DNA binding sites for Spl, a mammaliantranscription regulatory protein.

An eleven-base sequence (Table 2), which is conserved in both the RAmy3Dand the RAmy3E promoter, contains a single copy of the CGGC sequence. Atandem duplication of the CGGC sequence is also found in the promotersof the Taka-amylase genes of Aspergillus oryzae. These CGGC sequencesare found in the 87 base region of the Taka-amylase promoters (fromposition -377 to -290) that has been implicated in the metabolicregulation of these genes.

(b) General Sources of Promoter and Signal Sequences

In view of the guidance of the present disclosure and by using a numberof standard procedures, one of skill can identify suitable promoters andsignal sequences in addition to those described above for use in thisinvention. While the gene can be amplified directly from a mRNA extractusing PCR, the first step is generally to produce a genomic or cDNAlibrary.

In brief, genomic or cDNA libraries are prepared according to standardtechniques as described, for instance, in Sambrook, et al., MolecularCloning--A Laboratory Manual, Cold spring Harbor Laboratory, Cold SpringHarbor, N.Y. 1989. To construct genomic libraries, large segments ofgenomic DNA are generated by random fragmentation and are ligated withvector DNA to form concatemers that can be packaged into the appropriatevector. Two kinds of vectors are commonly used for this purpose,bacteriophage lambda vectors and cosmids. Alternatively, genomiclibraries can be purchased from commercial sources (e.g., Clontech, PaloAlto, Calif.).

In the present invention, cDNA libraries enriched, for example, forα-amylase secreting mRNA sequences are used to screen for the desiredgenes. Preparation of appropriately enriched cDNA would involve the useof plant organs over expressing and secreting α-amylase, e.g., aleuronelayers. Other organs would include roots, stems, leaves and panicles.Briefly, mRNA from select tissue is isolated and cDNA is prepared. Shortchains of oligo d-T nucleotides are hybridized with the poly-A tails ofthe mRNA and serve as a primer for the enzyme, reverse transcriptase,which synthesizes a complementary DNA (cDNA) strand.

The cDNA can be optionally enriched for the desired sequences usingsubtraction hybridization procedures by labelling the cDNA andhybridizing it with mRNA from tissue that does not express the desiredmRNA according to the procedures. Proc. Natl. Acad. Sci. U.S.A.81:2194-2198 (1984).

Unreacted cDNA is isolated and used to prepare a library for screening.To do this, a second DNA strand is synthesized using the first cDNAstrand as a template. Linkers are added to the double-stranded cDNA forinsertion into a plasmid or λ phage vector for propagation in E. coli.

Identification of clones harboring the desired sequences is performed byeither nucleic acid hybridization or immunological detection of theencoded protein, if an expression vector is used. Typically,oligonucleotide probes specific for the gene of interest are used.

After identification of a cDNA corresponding to the gene of interest,the cDNA molecule can be used as a probe of a genomic DNA, containingthe gene of interest. Corresponding genomic clones are identified.Genomic clones isolated with this approach will contain gene sequencesthat include the promoter, coding region, introns and terminators.

Alternatively, the probes specific to the gene of interest can be usedto directly probe genomic DNA libraries from a selected plant. Sequenceshomologous to the probe can be isolated by standard techniques,including hybridization screening or polymerase chain reaction.

Oligonucleotide and cDNA probes useful for identification of otherpromoters and signal sequences can also be prepared from conservedregions of related genes in other species. By comparing the nucleotidesequences of the known proteins, one simply identifies conservedsequences in the genes and uses those sequences as probes or as PCRprimers to locate homologous sequences in genomic or cDNA libraries ofother plants. A number of references compare regions of nucleotidehomology and amino acid identity regarding secreting genes and they areprovided below. Such conserved sequences can be used to isolate othergenes having a hormonal or other metabolite responsive promoter.

Probes, typically used to identify related but heretofore unknown targetsequences, can be hybridized under stringent conditions to ensure thatthe sequences are in fact related. Typically, stringent conditionssuitable for finding related sequences would be performing thehybridization at a melting temperature (Tm) of between -15° C. to -20°C.

(c) Cloning of the Desired DNA Sequences

Once the DNA encoding the desired sequences has been located, sufficientquantity of the gene must be generated to facilitate subsequentrecombinant manipulations. Although the sequences can be directlyamplified by PCR, they are most commonly replicated in an intermediatebacterial host. Most commonly in a bacteria of the genera Escherichia,Bacillus and Streptomyces. Cloning for amplification of intermediatevectors is most preferred in E. coli because that organism is easy toculture and more fully understood than other species of prokaryotes.

Sambrook, supra, contains methodology sufficient to conduct clonings inE. coli. Strain HB101 is a useful strain which is typically grown onLuria broth (LB) with glucose, Difco's Antibiotic Medium #2 and M9medium supplemented with glucose and acid-hydrolyzed casein amino acids.Strains with resistance to antibiotics are maintained at the drugconcentrations described in Sambrook, supra.

Transformations are performed according to the method described byMorrison, D. A. (1977), J. Bacteriol., 132:349-351; or by Clark-Curtiss,J. E. and Curtiss, R., 1983, in Methods in Enzymology, 101:347-362, Wu,R., Grossman, L. and Moldave, K., eds., Academic Press, New York.Representative vectors include pBR322 and the pUC series which areavailable from commercial sources.

4. Transcription and Translation Terminators

The expression cassettes or chimeric genes of the present inventiontypically have a transcriptional termination region at the opposite endfrom the transcription initiation regulatory region. The transcriptionaltermination region may normally be associated with the transcriptionalinitiation region or from a different gene. The transcriptionaltermination region may be selected, particularly for stability of themRNA to enhance expression. Illustrative transcriptional terminationregions include the NOS terminator from Agrobacterium Ti plasmid and therice α-amylase terminator.

Polyadenylation tails, Alber and Kawasaki, 1982, Mol. and Appl. Genet.1:419-434 are also commonly added to the expression cassette to optimizehigh levels of transcription and proper transcription termination,respectively. Polyadenylation sequences include but are not limited tothe Agrobacterium octopine synthetase signal, Gielen, et al., EMBO J.3:835-846, 1984 or the nopaline synthase of the same species Depicker,et al., Mol. Appl. Genet. 1:561-573, 1982.

Since the ultimate expression of the desired gene product will be in aeukaryotic cell (e.g., a member of the grass family), it is desirable todetermine whether any portion of the cloned gene contains sequenceswhich will be processed out as introns by the host's splicosomemachinery. If so, site-directed mutagenesis of the "intron" region maybe conducted to prevent losing a portion of the genetic message as afalse intron code, Reed and Maniatis, Cell 41:95-105, 1985.

5. Transformation of Plant Cells

(a) Direct Transformation

Vectors containing a chimeric gene of the present invention canintroduced into plant cells by a variety of techniques. These vectorsmay include selectable markers for use in plant cells (such as, thenptII kanamycin resistance gene). The vectors may also include sequencesthat allow their selection and propagation in a secondary host, such as,sequences containing an origin of replication and a selectable marker.Typical secondary hosts include bacteria and yeast. In one embodiment,the secondary host is Escherichia coli, the origin of replication is acolE1-type, and the selectable marker is a gene encoding ampicillinresistance. Such sequences are well known in the art and arecommercially available as well (e.g., Clontech, Palo Alto, Calif.;Stratagene, La Jolla, Calif.).

The vectors of the present invention may also be modified tointermediate plant transformation plasmids that contain a region ofhomology to an Agrobacterium tumefaciens vector, a T-DNA border regionfrom Agrobacterium tumefaciens, and chimeric genes or expressioncassettes (described above). Further, the vectors of the invention maycomprise a disarmed plant tumor inducing plasmid of Agrobacteriumtumefaciens.

Vectors useful in the practice of the present invention may bemicroinjected directly into plant cells by use of micropipettes tomechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet,202:179-185, 1985. The genetic material may also be transferred into theplant c/ell by using polyethylene glycol, Krens, et al., Nature, 296,72-74, 1982.

Another method of introduction of nucleic acid segments is high velocityballistic penetration by small particles with the nucleic acid eitherwithin the matrix of small beads or particles, or on the surface, Klein,et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta,185:330-336 (teaching particle bombardment of barley endosperm to createtransgenic barley).

Yet another method of introduction would be fusion of protoplasts withother entities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79,1859-1863, 1982.

The vector may also be introduced into the plant cells byelectroporation. (From, et al., Pro. Natl Acad. Sci. USA 82:5824, 1985).In this technique, plant protoplasts are electroporated in the presenceof plasmids containing the gene construct. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

(b) Vectored Transformation

A common vector method of introducing the vector into plant cells is toinfect a plant cell with Agrobacterium tumefaciens previouslytransformed with the gene. Under appropriate conditions known in theart, the transformed plant cells are grown to form shoots or roots, anddevelop further into plants.

Agrobacterium a representative genus of the gram-negative familyRhizobiaceae. Its species are responsible for plant tumors such as crowngall and hairy root disease. In the dedifferentiated tissuecharacteristic of the tumors, amino acid derivatives known as opines areproduced and catabolized. The bacterial genes responsible for expressionof opines are a convenient source of control elements for chimericexpression cassettes.

Heterologous genetic sequences, such as the chimeric genes of thepresent invention, can be introduced into appropriate plant cells, bymeans of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid istransmitted to plant cells on infection by Agrobacterium tumefaciens,and is stably integrated into the plant genome. J. Schell, Science 237:1176-1183, 1987.

Ti plasmids contain two regions essential for the production oftransformed cells. One of these, named transferred DNA (T-DNA), istransferred to plant nuclei and induces tumor formation. The other,termed virulence region, is essential for the transfer of this T-DNA butis not itself transferred. The transferred DNA region, which transfersto the plant genome, can be increased in size by the insertion of thegene encoding group 3 LEA proteins without its ability to be transferredbeing affected.

A modified Ti plasmid, in which the tumor-causing genes have beendeleted, can be used as a vector for the transfer of the gene constructsof this invention into an appropriate plant cell.

Construction of recombinant Ti plasmids in general follows methodstypically used with the more common bacterial vectors such as pBR322.Additional use can be made of accessory genetic elements sometimes foundwith the native plasmids and sometimes constructed from foreignsequences. These may include but are not limited to "shuttle vectors",Ruvkun and Ausubel, 1981, Nature 298:85-88, promoters, Lawton, et al.,1987, Plant Mol. Biol. 9:315-324 and structural genes for antibioticresistance as a selection factor, Fraley, et al., Proc. Natl. Acad. Sci.80:4803-4807, 1983.

Species which are a natural plant host for Agrobacterium may betransformable in vitro. Monocotyledonous plants, and in particular,cereals and grasses, are not natural hosts to Agrobacterium. Attempts totransform them using Agrobacterium have been unsuccessful untilrecently. Hooykas-Van Slogteren, et al., Nature 311:763-764, 1984. Thereis growing evidence now that certain monocots can be transformed byAgrobacterium. Using novel experimental approaches that have now becomeavailable, cereal and grass species may now be transformed.

Promoters directing expression of selectable markers used for planttransformation (e.g., nptII) should operate effectively in plant hosts.One such promoter is the nos promoter from native Ti plasmids,Herrera-Estrella, et al., Nature 303:209-213, 1983. Others include the35S and 19S promoters of cauliflower mosaic virus, Odell, et al., Nature313:810-812, 1985, and the 2' promoter, Velten, et al., EMBO J. 3,2723-2730, 1984.

6. Plant Regeneration

After determination of the presence and expression of the desired geneproducts, whole plant regeneration is desired. Plant regeneration fromcultured protoplasts is described in Evans, et al., Handbook of PlantCell Cultures, Vol. 1: (MacMillan Publishing Co. New York, 1983); andVasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants,Acad. Press, Orlando, Vol. I, 1984, and Vol. III, 1986.

All plants from which protoplasts can be isolated and cultured to givewhole regenerated plants can be transformed by the present invention sothat whole plants are recovered which contain the transferred gene. Itis known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to all major species ofsugarcane, sugar beet, cotton, fruit and other trees, legumes andvegetables, and monocots.

Some suitable plants include, for example, species of wheat, rice, oats,rye, corn, sorghum, millet and barley.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts containing copies ofthe heterologous gene is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.

Alternatively, embryo formation can be induced from the protoplastsuspension. These embryos germinate as natural embryos to form plants.The culture media will generally contain various amino acids andhormones, such as auxin and cytokinins. It is also advantageous to addglutamic acid and proline to the medium, especially for such species ascorn and alfalfa. Shoots and roots normally develop simultaneously.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is fully reproducible and repeatable.

The mature plants, grown from the transformed plant cells, are selfedand non-segregating, homozygous transgenic plants are identified. Theinbred plant produces seed containing the chimeric gene of the presentinvention. These seeds can be malted to produce the protein orpolypeptide of interest.

The inbreds according to this invention can be used to develop hybridsor novel varieties embodying the desired traits. Such plants would bedeveloped using traditional selection type breeding.

The transgenic seeds produced by the method of the present invention canbe formed into seed products.

B. Antisense Applications

In addition to the above indicated genes, one may also have constructswhich provide for inactivation of endogenously expressed genes. Ofparticular interest is the inactivation of genes that are expressedduring germination and seedling elongation. These genes may include oneor more of the amylases, e.g., RAmy3B, RAmy3C, RAmy3E or homologsthereof.

Inactivation of gene expression may be achieved in a number of ways. Themost convenient is the use of an anti-sense sequence, where theanti-sense sequence may be complementary to any portion of the mRNA,including both the non-coding and coding regions. Normally, theanti-sense sequence will be at least about 30 nt, more usually at leastabout 50 nt, and may be up to or greater than the sequence of the mRNAto which the anti-sense sequence is complementary.

In one embodiment, the 3'-terminal sequence of the anti-sense sequenceis selected to provide for mRNA stability, there being a number ofsequences which are known to destabilize the mRNA which can be avoided.

The transcription initiation region for the anti-sense sequence may beconstitutive or inducible. A relatively strong promoter may be employed,such as, the 35S CMV promotor, the RUBSICO promoter, or beta-conglycininpromoter. Preferably, the transcription initiation region is inducibleso as to be induced during the malting process. To enhance thetranscription of the anti-sense sequence, one may use various enhancersassociated with other promoters to increase the rate of transcription ofthe anti-sense sequence.

It is not necessary that all expression of one or more proteinsnaturally produced during malting is inhibited, it being sufficient thatthere be at least about a 10%, preferably at least about a 25% reductionin expression, so as to increase the proportion of the desired proteinin the malting product. Enhancers which find use include the 35S CMVenhancer, and the introns of the alcohol dehydrogenase gene of maize.

III. The Malting Process

The malting process is a multi-step process. The first step is steeping.During steeping seed is immersed in or sprayed with water to increasethe moisture content of the seed to between 35-45%. This initiatesgermination. Steeping typically takes place in a steep tank which istypically fitted with a conical end to allow the seed to flow freelyout. The addition of compressed air to oxygenate the steeping process isan option. The temperature is controlled at approximately 22° C.depending on the seed.

After steeping, the seed is transferred to germination compartments. Theseed is either wet or dry transferred. The germination bin contains airsaturated with water and is under controlled temperature and air flows.The typical temperatures are between 12°-25° C. and germination ispermitted to continue for from 3 to 7 days.

Where the heterologous protein is operably linked to a induciblepromoter requiring a metabolite such as sugar or plant hormone, thismetabolite is added, removed or depleted from the steeping water mediumand/or is added to the water saturated air used during germination. Theseed absorbs the aqueous medium and begins to germinate expressing theheterologous protein. The medium may then be withdrawn and the maltingbegun, by maintaining the seeds in a moist temperature controlledaerated environment. In this way, the seeds may begin growth prior toexpression, so that the expressed product is less likely to be partiallydegraded or denatured during the process. Other components included inthe imbibition medium may be plant hormones, such as gibberellic acid,generally in an amount from about 2.5 to 100 μM.

Where the promoter is induced by sugar, glucose or sucrose can be addedto the imbibition media during steeping or during germination. The sugarconcentration may range up to about 12 weight percent of the medium.

More specifically, the temperature during the imbibition or steepingphase will be maintained in the range of about 15°-25° C., while thetemperature during the germination will usually be about 20° C. The timefor the imbibition will usually be from about 2 to 4 days, while thegermination time will usually be an additional 2 to 10 days, moreusually 3 to 7 days. Usually, the time for the malting does not exceedabout ten days. The period for the malting can be reduced by using planthormones during the imbibition, particularly gibberellic acid.

Germinated seed produced by this method may be used to producegerminated seed products.

To achieve maximum production of recombinant protein from malting, themalting procedure will be modified to accommodate de-hulled andde-embryonated seeds. The hulls and embryos are dehulled andde-embryonated using standard means which include rollers, othermechanical means of breaking the intact embryos free of hull andendosperm. Screening is typically used to separate the embryos fromunwanted seed debris. Isolated transgenic embryos are germinated insteeping water containing CaCl₂ (approx 10 mM).

In the absence of sugars from the endosperm, there is expected to be a 5to 10 fold increase in RAmy3D promoter activity and thus expression ofthe heterologous protein. Alternatively when embryoless-half seeds areincubated in 10 mM CaCl₂ and 5 μm gibberellic acid, there is a 50 foldincrease in RAmy1A promoter activity.

In this system, recombinant proteins under the control of RAmy1A (HV18or other homologs) and RAmy3D promoters are secreted into the medium.Specialized malting bins or steep tanks may be used. The embryos andembryoless-half seeds are mechanically disrupted to release any secretedprotein between cells and tissues. The mixture is suspended in a buffersolution to retrieve soluble proteins. Conventional protein isolationand purification methods are then used to purify the recombinantprotein. Parameters of time, temperature pH, oxygen, and volumes areadjusted through routine methods to optimize expression and recovery ofheterologous protein.

An optional step is kilning the germinated seed. Kilning is a lowtemperature drying procedure that reduces moisture concentration to4-6%. Temperatures during kilning are between 40°-85° C. Typically thelower temperatures of less than 60° C. are used until the seed have amoisture content of between about 10 and 20%. Final drying is at highertemperatures of above 60° C.

The kiln-dried material (or malted seeds) may then be used directly, forexample, where the recombinant protein is useful in veterinaryapplications (e.g., animal feed containing a an increased protein value,growth hormone or vaccine). The seed may be formulated into a mashproduct.

The mash may also be processed by mechanical disruption of the seeds andbringing the total protein into solution. The cellular debris may thenbe separated by any convenient means, such as settling, centrifugation,filtration, or the like. The supernatant or filtrate will normallyinclude from 1 to 40 weight percent of the desired product of totalprotein in the medium, preferably at least about 30 weight percent.

Where the desired product is not water soluble, one may need to extractthe desired product with a convenient solvent or use another processwhich allows for solubilization and/or extraction of the product withoutloss of the desired activity of the product or which allows forrenaturation.

After isolation of the protein of interest from the aqueous medium, onemay then purify the product in accordance with conventional ways. Sincethe product will be a substantial portion of the total protein presentin the mixture, frequently being present in the greatest percentage ofany individual protein, purification is greatly simplified. Furthermore,contaminants in the product after purification are not likely to be ofphysiological concern for many of the applications of the products,including therapeutic applications.

By providing for malting, seeds can be germinated under conditions wherea desired product can be produced in the germinated seeds to provide fora high propc on of the total protein in the malting mash being thedesired protein. By breaking the cells, separating the cellular debrisfrom the protein, and isolating the supernatant from the mash, theprotein may be easily isolated and purified, being a major component ofthe total protein in the medium.

As distinct from other methods of producing proteins, the subject methodprovides for high levels of economic production of proteins in a crudeform which can be easily purified. The system lacks the potential forthe production of endo and exotoxins, which is of concern withprokaryotes. The system allows for storage under ambient conditionswithout significant loss of seed viability or product loss. The productcan be produced on demand. In this way, proteins can be produced inaccordance with need, where the source of the protein can beconveniently and safely stored.

In one embodiment, the malted transgenic seeds of the present invention,containing a protein of interest, can be used as a malted seed feedproduct for animals. The malted transgenic seeds can be mixed withnormal (i.e., non-transgenic) seeds to obtain the desired concentrationof the protein of interest in an animal feed product.

The following is an example of protein production by the malting processof the present invention. Varieties of rice containing the human genefor α-1-antitrypsin (i.e., transgenic rice) are inbred to producehomozygous lines. These transgenic lines are cultivated in the field andmature seeds are harvested using conventional agricultural practices.

The transgenic seeds are transported to a malting facility (malthouse)where they are soaked (steeped) in water for 48 hours. The seeds arethen transferred to malting bins where they are allowed to germinatefrom 2 to 4 days. During germination, the temperature and humidity ofthe seed bed is monitored to ensure ideal germination conditions. Insome cases, chemicals such as the plant hormone, gibberellic acid, maybe added to enhance the expression of the α-1-antitrypsin gene which isunder the control of a gibberellic acid inducible promoter (e.g.,RAmy1A, RAm3C, HV18).

After optimum germination and expression of the α-1-antitrypsin genehave been achieved, the seeds are mashed (for example, by gentlegrinding) to disrupt tissues and remove the hulls from the seeds. Theseed mash is suspended in a protein extraction buffer. Such a buffertypically contains protease inhibitors, reducing agents and a bufferingagent (such as, "TRIS" or sodium or potassium phosphate).

The mash is agitated or stirred to ensure that all secreted protein isfreed from tissues and cells. Large particulate matter, such as hull,plant tissues, and other debris are then removed by filtration orcentrifugation. The supernatant is collected and chilled to reduceproteolysis of α-1-antitrypsin.

The supernatant is subjected to various purification schemes used in thewet-milling industry (e.g., hydrocloning and ion exclusionchromatography) to remove un-wanted proteins and to concentrateα-1-antitrypsin. Alternatively, ammonium sulfate precipitation can alsobe used to concentrate the α-1-antitrypsin.

Affinity- and ion-exchange chromatography can be used to purify theα-1-antitrypsin away from other proteins in the supernatant. Thepresence of α-1-antitrypsin in the various chromatographic fractions canbe detected using standard photometric assays.

In another embodiment, after the transgenic seeds are transported to amalting facility (malthouse) they are dehulled and deembryonated (i.e.,mechanical separation of the embryos and endosperm portions of theseed). The embryos and endosperms are separately soaked (steeped) inwater for 48 hours. The seeds are treated as described above. Theseparated embryos are treated as follows. Expression of the RAmy3Dpromoters is induced in the absence of sugar and/or by the addition ofchemicals, such as a plant hormone, e.g., abscisic acid.

After optimum germination and expression of the α-1-antitrypsin genehave been achieved, the embryo and endosperm portions are mixed and thenmashed (i.e., gentle grinding) to disrupt seed tissues. The mash is thentreated as above for purification of the α-1-antitrypsin polypeptide.

IV. Production of Recombinant Proteins Using a Cell Culture Process.

In the second aspect, the invention relates to the regulated expressionof recombinant proteins in cereal cell culture. In one embodiment of theinvention, the cells are derived from scutellar epithelium of cerealplants. The chimeric genes, vectors and methods described above may beimplemented in the practice of this aspect of the invention. In thisaspect, the invention includes modulating expression of a polypeptide inmonocot plant tissue cell culture. Transgenic plant cells are producedthat contain a chimeric gene having at least the following components:

(i) a transcription regulatory region inducible during seed germination,where expression mediated by said region is specifically regulatable bya small molecule. Several examples of such regions and small moleculeregulators are described below; and

(ii) a heterologous DNA sequence that encodes the polypeptide, wheresaid DNA sequence is operably linked to said promoter. The chimeric genemay also contain a signal sequence to facilitate secretion from the cellof the polypeptide encoded by the heterologous DNA sequence.

The transgenic cells are cultured under conditions that facilitate plantcell growth. Expression of the polypeptide of interest is modulated byaddition or removal of at least one small molecule to the plant cellculture.

For example, the principle of using different cereal α-amylase promotersto express a recombinant protein in tissue culture cells is illustratedin FIG. 1A. In this figure, the sugar-repressible promoter for the riceα-amylase gene, RAmy3D, was used to express the bacterial reporter gene,gusA, in rice. The gusA gene encodes the enzyme, beta-glucuronidase(GUS), that produces a blue chromophore in tissues expressing the gene.This chromophore can be easily detected using a histochemical stainingmethod. As can been seen in this figure, the product of gusA isrepressed in rice cells when the culture medium contains 3% sugar.

Cells or tissues derived from cereal plants can be transformed singly ortogether (i.e., co-transformation) with the expression constructsdescribed above. Once integrated into the plant genome, the recombinantprotein can be recovered and purified from the medium of culturedtransgenic cells.

The vectors of this invention can be used to facilitate the expressionand/or secretion of heterologous protein in cell culture. The plantcells are placed and maintained into suspension culture and inducedthrough the variety of inducers described above to produce high levelsof the desired heterologous protein. The protein is then isolated usingconventional technology.

Because the purifications are dramatically varied for individualproteins, it is sufficient to indicate that the initial purificationprocess will typically follow the purification process of the nativeprotein from its host. Because the growth media of the plant suspensionculture, as used in the present invention, is typically more simple thanthe normal host environment of the protein of interest, the purificationprocedures may be appropriately modified and simplified by those ofskill in the art.

It is evident from the above results, that plant cells can be engineeredand the cells used to propagate plants. The plant cells can be modifiedto provide for expression constructs that allow controlled expression ofthe coding sequence in the construct to provide the expression productas the major product.

By combining the technology of the present invention withwell-established production methods (e.g., plant cell fermentation, cropcultivation, and product recovery), recombinant protein can beefficiently and economically produced for the biopharmaceutical,industrial processing, animal health and bioremediation industries.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters which could be changed or modified to yieldessentially similar results.

EXAMPLES

General Methods

Generally, the nomenclature and laboratory procedures with respect tostandard recombinant DNA technology can be found in Sambrook, et al.,Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. 1989 and in S. B. Gelvin and R. A. Schilperoot,Plant Molecular Biology, 1988. Other general references are providedthroughout this document. The procedures therein are known in the artand are provided for the convenience of the reader.

Example 1 Expression of β-glucuronidase Under the Control of theα-amylase Promoter in Rice Cell Culture

A. Initiation of Scutellar Callus and Suspension Cultures.

Rice seeds (Oryza sativa L. cv. M202) were provided by Dr. M. Brandon(California Rice Experimental Station). Seeds were dehulled, washedthree times with water, rinsed in 70% ethanol for 20 sec and thensurface-sterilized in 1% sodium hypochlorite with a few drops of Tween20 under vacuum for 20 min. Sterilized seeds were washed three timeswith sterile distilled water. Seven seeds were placed in 15 cm petridishes containing LS medium with 2 mg/l 2,4-D and 30 g/l sucrose. Theseeds were incubated in the dark at 28° C. and checked periodically tomonitor the growth of scutellar-derived callus. Callus formation fromscutellum tissue and/or embryo was visible after 5 days. After 30 to 40days, clumps of friable calli, about 1 cm in diameter, were saved andthe remaining tissue was discarded.

To initiate a suspension culture, friable calli were gently agitated ina petri dish with liquid AA medium as described by Thompson J A,Abdullah R and Cocking E C, Protoplast culture of rice (Oryza sativa L.)using media solidified with agarose (Plant Sci. 47:123-133 (1986) toreduce the calli to small clusters of cells. Cell clusters from about20-30 clumps of calli were then transferred to a 125 ml Erlenmeyer flaskand the liquid was replaced with 25 ml of fresh AA medium. The flaskswere incubated in the dark on a rotary platform shaker at 110 rpm and28° C. The primary culture was sub-cultured every 4 to 5 days withrepeated screening for small cell clusters. This was accomplished bypassing the culture sequentially through nylon filters of 1000 μm and500 μm pore size. After two months of subculture, a finely divided andrapidly growing suspension culture was obtained. This culture wassubsequently maintained by weekly subculture in AA medium containing 3%sucrose.

B. Construction of the RAmy3D/GUS Gene Fusion

The RAmy3D promoter/GUS gene fusion shown in FIG. 2 was constructed inthree steps. First, a 1.5 kb Sall fragment containing the promoter andpart of the coding region from rice genomic clone λOSglA as described byHuang, et al., 1990a, Nucleic Acid Res., 18:7007-7014 was subcloned intopBluescript KS- to produce the plasmid plAS1.5. The Alul fragment fromplAS1.5 containing 876 bp of promoter and 66 bp of 5' untranslatedregion was subcloned into the EcoRV site of "PBLUESCRIPT KS+" to formplAlu.

Second, a plasmid containing a promoterless GUS cassette was constructedby subcloning the HindIII/EcoRI GUS cassette from pBl101 (Jefferson R A,1987, Assaying chimeric genes in plants: the GUS gene fusion system.Plant Mol. Biol. Reporter, 5:387-405) into pUC19 to form pBl201. A pUC19polylinker in front of the GUS coding region provides convenient cloningsites for inserting promoter fragments. Third, the RAmy3D promoterfragment was inserted into the promoterless GUS plasmid to produce theplasmid p3DG. The Xbal/Alul (in HindIII site) promoter fragment fromplAlu was ligated into Xbal/Smal digested pBl201. The final 11 bp ofRAmy3D 5' untranslated region was substituted by 21 bp from thepolylinker resulting in the 5.83 kb plasmid, p3DG.

The junction between the RAmy3D promoter and the 5' end of the GUS genewas confirmed by DNA sequencing. DNA restriction digest, DNA gelelectrophoresis, ligation, transformation, plasmid DNA isolation and DNAsequencing followed standard procedures (Sambrook, et al., MolecularCloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. 1989.

C. Protoplast isolation and DNA transformation

Three days after subculture, a 60 ml of rice suspension culture wasgiven a ten minute, 45° C. heat-treatment and transferred to two glasscrystallizing-dishes. The AA medium was removed and the cells in eachdish were mixed with the 20 ml of enzyme mixture (1% Cellulose RS, 1%Macerozyme R10 in CPW medium described by Thompson, et al., 1986, PlantSci. 47:123-133. The cell walls were digested at 28° C. for 15 hourswhile shaking at 45 rpm.

After digestion, the protoplasts were screened through 150, 50 and 20 μmnylon filters, washed three times by centrifuging for 10 min at 80 g,and gently resuspended in 40 ml of CPW medium. Then the suspension wasadjusted to 5 million protoplasts/ml.

Two and one-half million protoplasts in 0.5 ml volume were mixed with 5μg of p3D2 DNA, 25 μg of calf thymus carrier DNA and 5 μg of pGL2plasmid DNA carrying the CaMV 35S promoter/hph gene fusion encodinghygromycin-resistance as described by Shimamoto K, Terada R, Izawa T andFujimoto H (Fertile transgenic rice plants regenerated from transformedprotoplasts Nature 338:274-276 (1989)), transferred to a cuvette, placedon ice for ten min, then electroporated with a "GENE PULSER"electroporator (Bio-Rad)(300 volt/cm, 560 mF and 600 Ohms).

After being kept on ice for an additional 10 min, the protoplasts weremixed with 0.5 ml of 4× KPR medium (Thompson, et al., Supra 1986) and 1ml of melted 2.4% "SEA PLAQUE" low gelling agarose, plated in Petridishes and incubated at 28° C. in the dark.

Ten days after plating, the agarose in each Petri dish was separatedinto four pieces and transferred into a 6 cm Petri dishes containing 5ml of liquid KPR medium. Four days later hygromycin was added to eachdish to a final concentration of 50 mg/ml. Hygromycin-resistant colonieswere picked and grown in liquid AA medium to form a number of celllines. A sample from each cell line was then assayed for GUS activity bystaining the cells with X-glu (Jefferson, et al., supra 1987). Celllines having GUS activity were retained. Uniform cell lines wereobtained by two additional rounds of isolating cell single cell clusterscoupled with selection for GUS expression. Integration of p3DG DNA intothe rice genome was verified by Southern blot analysis.

D. RNA Isolation and Dot Blot Hybridization

Total RNA was isolated from cell suspension culture using a modificationof the phenol/SDS procedure Ausubel F M, Brent R, Kingston R E, Moore DD, Seidman J G, Smith J A and Struhl K: Current Protocols in MolecularBiology. (1989). Approximately one gram of cultured rice cells wereground into a fine powder with sand and 5 ml of liquid nitrogen untilall the liquid nitrogen evaporated. Then 2.5 ml of TLE buffer (0.2M TrispH. 8.2, 0.1M LiCl, 5 mM EDTA, 20 mM sodium metabisulfite) was added andgrinding continued until the sample was completely liquefied.

At this point, 0.5 ml of 10% SDS, 2.5 ml of phenol and 2.5 ml ofchloroform were added to the mortar sequentially and mixed well bygrinding. The sample was centrifuged at 4000 g for 15 min and theaqueous phase removed and extracted with chloroform. Total RNA wasprecipitated by the addition of 1/3 volume of 8M LiCl and the mixturewas allowed to sit overnight at 4° C. The RNA was harvested bycentrifugation at 16,000 g for 30 min at 4° C. and the RNA pellet wasdissolved in 0.5 ml of double distilled water treated withdiethylpyrocarbonate. The RNA was extracted once more with chloroformand then precipitated with ethanol. The RNA yield was approximately 500μg from each gram (fresh weight) of cells.

The pre-hybridization and hybridization of α-amylase probe to themembrane under α-amylase group-specific conditions was as previouslydescribed by Huang N, Koizumi N, Reinl S and Rodriguez R L (Structuralorganization and differential expression of rice α-amylase genes NucleicAcids Res. 18:7007-7014 (1990a) and Huang N, Sutliff T D, Litts J C andRodriguez R L: Classification and characterization of rice α-amylasemultigene family. Plant Mol. Biol. 14:655-668 (1990b)).

Four different rice α-amylase genes were used as probes. The RAmy1Aprobe, a 1.6 kb Xbal fragment from pOS103 described in O'Neill S D,Kumagai M H, Majumdar A, Huang N, Sutliff T D and Rodriguez R L (Theα-amylase genes in Oryza sativa: characterization of cDNA clones andmRNA expression during seed germination. Mol. Gen. Genet. 221:235-244(1990)) cross hybridized with the closely related genes RAmy1B andRAmy3C under the stringency conditions used.

The RAmy3D probe, a 1.6 kb Xbal fragment from pOS137 O'Neill, supra! wasused under gene-specific conditions. The RAmy3A probe, a 3.5 kb EcoRIfragment from λOS7D (Sutliff T D, Huang N, Litts J C and Rodriguez R L:Characterization of an α-amylase multigene cluster in rice. PlantMolecular Biology. 16:579-591 (1991)) was used under highly stringent,gene-specific conditions. The RAmy3E probe, a 2 kb HindIII fragmentincluding the two introns exons II and III, and the 3' end as describedby Huang N, Koizumi N, Reinl S and Rodriguez R L (Structuralorganization and differential expression of rice α-amylase genes.Nucleic Acids Res. 18:7007-7014 (1990a)).

E. DNA Isolation and Southern Blot Hybridization

Total genomic DNA was isolated using a small scale CTAB proceduredescribed by Rogers SO and Bendich A J (Extraction of DNA from milligramamounts of fresh, herbarium and mummified plant tissues. Plant Mol.Biol. 5:69-76 (1985)). Southern blot analysis of transformed andun-transformed cultured cells was the same as previously described inHuang N, Sutliff T D, Litts J C and Rodriguez R L (Classification andcharacterization of rice α-amylase multigene family. Plant Mol. Biol.14:655-668 (1990b)).

The DNA probes used were the HindIII/EcoRI fragment of pBI201(Jefferson, Supra) for the GUS gene and pOS103 as described in O'Neill SD, Kumagai M H, Majumdar A, Huang N, Sutliff T D and Rodriguez R L (Theα-amylase genes in Oryza sativa: characterization of cDNA clones andmRNA expression during seed germination. Mol. Gen. Genet. 221:235-244(1990) for rice α-amylase gene RAmy1A.)

F. Isolation of Total Protein and β-glucuronidase (GUS) Activity Assay

Total water soluble protein was isolated from suspension culture cellsbased on the procedure described by Jefferson, supra. A 200 mg sample ofsuspension culture cells was ground in a mortar and pestle for one minin the presence of sand and 0.5 ml of GUS extraction buffer (Jefferson,supra). The slurry was transferred to a 1.5 ml microfuge tube and thecell debris removed after 5 min of centrifugation at room temperature.The supernatant was saved as a crude extract of water soluble protein.The GUS activity was measured by the fluorometric assay procedure(Jefferson supra) The background level of GUS activity in control,untransformed cells was negligible.

GUS activity was also assayed in whole cultured cells by calorimetricmethods (Jefferson, supra). Fresh or frozen cultured cells were put ineither a 1.5 ml tube, a 3.5 cm Petri dish or a microtiter plate. Fivevolumes of sterile X-glu solution were added to the cells. The reactionwas incubated at 37° C. for 30 min. or longer.

G. Secretion of GUS Into the Media Using the RAmy1A Promoter inTransgenic Rice Cells.

The suspension cells are removed by filtration and the filtered media isassayed for GUS activity. The assay methods are as described above.

H. Results

1. Metabolic regulation of RAmy3D and RAmy3E in untransformed ricesuspension cultures.

The above experiments allow for the determination of which riceα-amylase gene(s) is metabolically regulated in rice cell cultures.Total RNA was isolated from a cell culture sampled over a period ofeight days. RNA dot blots were hybridized sequentially with fourdifferent α-amylase gene probes. The mRNA levels for the RAmy1A, RAmy1Band RAmy1C genes were low and did not change significantly during theeight day period. The level of RAmy3A mRNA was also low and showedlittle change during the culture cycle. The levels of RAmy3D and RAmy3EmRNA were low initially, but increased significantly after five days,reaching their peak levels at 8 and 6.5 days respectively. These resultsare consistent with previous studies which demonstrated that RAmy3D(Group 2) and RAmy3E (Group 5) mRNA were abundantly expressed in ricecell culture while the expression of RAmy1A/RAmy1B/RAmy1C (Group 1),RAmy2A (Group 4) and RAmy3A/RAmy3B/RAmy3C (Group 3) was either low orundetectable. Other workers have found moderate expression of RAmy1A andanother gene in the Ramy1 subfamily, but only in 14 day old cellcultures.

The concentration of sugar in the rice cell suspension culture mediumcorrelates with the amount of α-amylase enzyme produced by the cells. Toinvestigate this effect at the gene level, suspension cultures, normallymaintained in medium containing 3% sucrose, were subcultured into mediawith 1%, 3%, 6% or 12% sucrose. RNA was isolated from cells harvested atone day and at five days after subculture. RNA dot blots were hybridizedwith α-amylase gene probes. All cells harvested after one day hadapproximately the same levels of α-amylase gene expression, presumablybecause none of the cultures had yet depleted the sucrose from themedium.

The mRNA levels for the RAmy3D and RAmy3E genes increased significantlyafter five days in the culture with 1% sucrose medium, showing inductionof gene expression after the sugar was depleted from the medium.Cultures with higher initial sucrose concentrations still had only lowlevels of RAmy3D and RAmy3E gene expression after five days. The levelsof RAmy1A, RAmy1B, RAmy1C and RAmy3A gene expression changed little inany of the cultures.

Sucrose concentrations in the culture medium were altered to test theeffect on α-amylase gene repression. After four days of culture inmedium with an initial sucrose concentration of 3%, cultures weresubdivided and the sucrose concentration was increased to 6% and 12%.One of the cultures was washed and resuspended in sucrose-free medium(0%). Cultures were incubated for two more days and RNA was isolated foranalysis by slot blot hybridization.

RAmy1A, RAmy1B, RAmy1C and RAmy3A expression levels were consistentlylow in all subcultures. RAmy3D gene expression was significantly reducedin subcultures supplemented with 6% or 12% sucrose, relative to that ofthe sucrose-free subculture. RAmy3E gene expression remained high in alltreatments. Thus, within two days after the addition of sucrose to theculture media, RAmy3D was highly repressed while RAmy3E expression wasrelatively unchanged. It is not clear to what extent these results aredue to differential transcriptional control and/or differential mRNAstability.

2. Transformation of RAmy3D Promoter/GUS Into Rice Cell Lines.

A RAmy3D promoter/GUS gene fusion was constructed and used to transformrice protoplasts. The plasmid p3DG contains 876 bp of RAmy3D 5' flankingregion plus 66 bp of the 5' untranslated leader sequence linked to theGUS coding region (FIG. 2). Plasmid p3DG was introduced into riceprotoplasts by co-electroporation with the plasmid pGL2 which carriesthe hygromycin-resistance gene. Protoplast-derived colonies wereselected on hygromycin-containing medium and tested forco-transformation with the RAmy3D/GUS construct by staining a few cellsfrom each colony for GUS activity. Two cycles of hygromycin-resistanceselection and GUS activity screening were used to isolate the 3DG cellline.

DNA was isolated from the 3DG cell line and from a non-transformedcontrol cell line, digested with BamHI and subjected to Southern blothybridization (FIGS. 3A and 3B). When the blot was probed with the GUSgene, a strong hybridization signal to DNA from the 3DG cell line (lanes2-5, FIG. 3A) was observed. No hybridization was seen with DNA isolatedfrom the control cell line (panel GUS, lane 1 of FIG. 3A).

The negative result in the GUS panel (lane 1, FIG. 3A) was not due tothe lack of DNA transferred to the membrane. Equivalent amounts of DNAwere detected in all lanes when the same membrane was stripped of theGUS probe and rehybridized with a probe from the rice α-amylase geneRAmy1A (FIG. 3B). These bands hybridizing to the α-amylase probe havethe molecular weights predicted from the DNA sequence of the RAmy1Agene.

Using the GUS probe, the 3DG cell line had the same hybridizationpattern before (FIG. 3A, lane 2) and after (lane 4) the two cycles ofsingle cell clump selection, indicating that the plasmid DNA was stablyinherited as the cells proliferated.

Two types of structural evidence indicate that the RAmy3D/GUS DNA isintegrated into the chromosomes of the 3DG cell line. First, Southernblot analysis revealed that the GUS gene probe hybridized exclusively toundigested genomic DNA larger than the size of the p3DG plasmid (FIG.3A, lanes 3 & 5).

Second, digestion of the DNA from the 3DG cell line with endonucleaseBamHI resulted in multiple hybridization bands (FIG. 3A, lanes 2 & 4).BamHI does not cut within the RAmy3D/GUS gene construct, so each bandsize represents a different sized junction fragment between the uniqueBamHI site in the p3DG plasmid and a BamHI site in the adjacentchromosomal DNA.

Thus, multiple copies of the RAmy3D/GUS gene construct (and at least onecopy of the hygromycin-resistance gene) have been integrated into thegenome of the 3DG cell line. The low molecular weight bands and thefaint bands of hybridization on the Southern blot probably representfragments of the RAmy3D/GUS gene construct inserted into the ricegenome.

3. Metabolic Regulation of RAmy3D/GUS in Transgenic Rice Cell Lines.

Gene expression and enzyme activity for GUS was assayed in the 3DG cellline to determine whether the promoter fragment in the RAmy3D/GUSconstruct contains all of the cis-elements necessary for proper-expression and metabolic regulation of the gene. Dot-blot hybridizationusing a GUS gene probe indicated that the mRNA level from the GUS genein 3DG cells increased as sugar was depleted from the culture medium.

The GUS enzyme assay was used to test for the expression of RAmy3D/GUSin response to various concentrations of sucrose in the culture medium.The 3DG cell line was subcultured into modified AA medium containing 0%,3% or 12% sucrose. Three days later, water soluble protein was extractedand assayed for GUS by the fluorescence assay (FIG. 4). The GUS activityin cells cultured with no sucrose was 65-fold higher than that of cellsgrown in 3% sucrose and 130-fold higher than that of cells grown in 12%sucrose. Thus, the transcriptional activity of the RAmy3D promoter wasgreatly repressed in the presence of high levels of sucrose while beinghighly induced under conditions of sugar deprivation.

The timing of RAmy3D promoter induction in response to sugar deprivationwas studied by incubating 3DG cells in sucrose-free medium. There waslittle or no increase in GUS activity during the first eight hours ofincubation (FIG. 5). GUS activity increased rapidly between eight tothirty-two hours after sub culturing. The expression and metabolicregulation of the RAmy3D/GUS gene construct resembles that of theendogenous RAmy3D gene.

These results are similar to those of others who observed an increase intotal α-amylase mRNA beginning 4 hours after the start of sugardeprivation. Thus, the cis-element(s) responsible for metabolicregulation must be contained in the 942 bp promoter region on theRAmy3D/GUS construct.

The expression of the GUS gene product was visualized usinghistochemical staining methods (Jefferson, supra) as seen in FIG. 1Awhere cell cultures incubated in the absence of sugar show blue stainingwith relatively high blue staining evident and where 3% sugar repressingthe expression of the GUS gene product with relatively less bluestaining is evident.

4. Promoter Sequence Analysis

Promoter sequences for RAmy3D and RAmy3E were compared to gainadditional insight into the metabolic regulation of these genes. Tworegions of sequence similarity were previously identified in thepromoters of these genes. One of these regions consists of a 31 bpGC-rich sequence that is 71% identical between the RAmy3D and RAmy3Egenes. This sequence is not found in the RAmy1A promoter (FIG. 6A-6Cwhich compare the percent of G+C in the promoter regions of the 1A, 3Dand 3E promoters, respectively) or in any other rice α-amylase promoter.This sequence is found at position-264 in RAmy3D (FIG. 6D) and containsthree nearly perfect repeats of a hexanucleotide sequence composedsolely of G and C residues.

The RAmy3E promoter contains one complete and one partial copy of thehexanucleotide repeat sequence. The tandem duplication of GC-richhexanucleotides in the 31 bp GC-rich sequences is reminiscent of bindingsites for the mammalian transcription factor Spl.

An 11 bp sequence containing part of the GC-rich hexanucleotide is alsofound in the RAmy3D and RAmy3E promoters (FIG. 6D). These sequences mayrepresent cis-acting elements involved in the metabolic regulation ofthe rice α-amylase genes. GC-rich promoter sequences have also beenidentified in the metabolically regulated α-amylase genes of Aspergillusoryzae.

Example 2 Secretion of Heterologous Protein Across the Aleurone Layer ofan Intact Rice Seed Using the RAmy1A Promoter

A. Plasmids

Plasmids were constructed using standard recombinant DNA methods(Ausubel, et al., 1989, Current Protocols in Molecular Biology, NY JohnWiley and Sons and Sambrook, et al., supra, 1989). The RAmy1A gene ofrice was chosen because of its responsiveness to GA (O'Neil, et al.,1990 Mol. Gen Genet., 221, 235-224) and because it is the most active ofthe α-amylase genes expressed during seed germination (Karrer, et al.,1991, Plant Mol. Biol., 16, 797-805).

Two regions of the RAmy1A promoter were fused to the gus A reporter geneto produce plasmids pH4/GUS (-748 to +31) and pE4/GUS (-232 to +31).Both promoter regions contain three conserved sequences (⁻²¹⁴ CCTTTT⁻209, ⁻ 147TAACAAA⁻¹⁴¹, and ⁻¹³⁰ TATCCAT⁻¹²⁴) found in all theGA-responsive cereal α-amylase genes examined to date (Huang, et al.,1990). An additional pyrimidine box is present in pH4/GUS atposition-312.

The promoter for the RAmy1A gene was subcloned as a 2.3 kb DNA fragmentfrom the rice genomic DNA clone (lOSg2) into "pBLUESCRIPT M13+KS." Thenucleotide sequence of this promoter has been described in Huang, etal., 1990 Nuc. Acids Res. 18:7007-7014. The principal features of theseconstructs consists of the b-glucuronidase gene (gusA), together withthe transcriptional terminator of the nopaline synthase gene from pBI101as reported in Jefferson, 1987 EMBO Journal 6:3901-3907.

The expression cassette was inserted into the Smal site of "pBLUESCRIPT"and designated pBSGUS. RAmy1A(promoter)/gusA gene fusions wereconstructed by inserting restriction fragments containing the RAmy1Apromoter into pBSGUS. The restriction fragments used to make constructswere the PstI-HindIII fragment (-748 to +31, pH4/GUS) and the Psfl-EcoRIfragment (-232 to +31, pE4/GUS). The coordinates used to describe theserestriction fragments are based on transcription start point for RAmy1A(Huang, et al., supra 1990).

B. Rice Transformation

RAmy1A/GUS plasmids were co-transformed into rice protoplasts (Oryzasativa L. japonica varieties, Nipponbare, Kinuhikari and Toride-1) byelectroporation as previously described (Shimamoto, et al., 1989,Fertile transgenic rice plants regenerated from transformed protoplasts,Nature, 338:274-276 and Kyozuka and Shimamoto, 1991 Transformation andregeneration of rice protoplasts, in Plant tissue Culture Manual(Lindsey, K. ed). Dordrecht:Kluwer Academic Publishers B1:1-16).

The hph gene (hygromycin phosphotransferase) was used as the selectablemarker in these studies. Hygromycin B resistant calli were screened forGUS activity by incubating a portions of calli with X-glucuronidesolution (Jefferson, 1987 supra). GUS positive calli were furthercultured and plants were regenerated from these callus cultures.

C. Southern Blot Analysis

The GUS positive R1 plants, derived from two lines each of H4/GUS andE4/GUS primary transgenic lines, were used for Southern blot analysis.Total genomic DNA was isolated from mature leaves, digested by therestriction enzyme EcoRI and transferred onto a positively charged nylonmembrane (Amersham). The coding region of the gusA gene was labeled andamplified with digoxigenin11-dUTP by polymerase chain reaction and usedfor probing the intact RAmy1A/gusA genes. Hybridization andchemiluminescence signal detection were performed according tomanufacturer's specifications (Boeringer Mannheim Biochemica).

D. GUS Assays

For histochemical analysis of GUS activity, germinating seeds werehand-cut with a razor and stained with X-glucuronide solution(5-bromo-4-chloro-3-indolyl glucuronide) as previously described inKyozuka, et al., 1991 supra and Terada, et al., 1993, Plant J.3:2412-252. For quantitative analysis, crude extracts from transgenicrice seeds were used for fluorometric assays of GUS activity asdescribed previously (Kyozuka, et al., 1991; Terada, et al., 1993).

For developmental studies, transgenic R1 seeds were pealed off,sterilized with 10% NaOCl for 10 min and washed with distilled water.Seeds were germinated in plastic wells containing water for 2, 4, 6, and8 days at 30° C. under light.

For quantitative measurements of GUS activity germinating seeds weredivided into the embryo and endosperm portions. In the case of theembryo, residual amount of endosperm, roots and shoots were removedbefore the assay.

For the analysis of hormonal regulation of the RAmy1A/gusA chimericgenes, transgenic R1 seeds were deembryonated and the embryoless seedsand sliced longitudinally into three pieces. Each slice was treated withacetate buffer (10 mM sodium acetate pH 5.2), 10⁻⁷ M GA3 in acetatebuffer, 10⁻⁷ M GA₃ and 10⁻⁵ M ABA in acetate buffer, for 4 days at 30Cin the dark. Treatment slices were then used for the histochemical andthe quantitative GUS assays.

E. Results

1. Southern Blot Analysis of Transgenic Rice Plants

Southern blot analysis confirmed the presence of H4/GUS and E4/GUS genefusions in the rice genome using the coding region of gusA as a probe(FIG. 7A). The results indicated that complete sequences of the H4/GUSand E4/GUS chimeric genes were present in transgenic plants and that thegusA gene was stably transmitted to the progeny (FIGS. 7B and 7C). Inaddition to the complete copies of the transgenes, several rearrangedcopies were also detected. The copy number of intact chimeric genes wasestimated to be 1-3 per haploid genome.

2. GUS Activity in Transgenic Rice Seeds

To compare the relative expression levels of the H4/GUS and E4/GUSgenes, the embryos and endosperms of germinated transgenic seeds wereseparated and the GUS activity in each tissue was measuredfluorometrically (FIGS. 8A and 8B). GUS assays of endosperm tissue wereperformed at 6 days of germination, the time when α-amylase expressionin the aleurone layer is at its highest (FIGS. 9A-9D).

Histochemical examination of 6-day germinated seeds showed that GUSactivity was restricted to the scutellum of the embryo and the aleuronelayer of the endosperm. In all four lines transformed with the H4/GUSgene, the aleurone activity (partially shaded bars) was higher than thescutellum activity (cross-hatched bars) (FIG. 8A). Similarly, four ofthe five lines transformed with E4/GUS gene showed GUS activity to behigher in the aleurone than in the scutellum (FIG. 8B). Comparisonsbetween H4/GUS and E4/GUS transformed lines, revealed no significantdifferences in GUS activity in the scutellar and aleurone tissues.

3. Temporal and Spatial Regulation of RAmy1A/GUS Expression DuringGermination of Transgenic Rice Seeds

To investigate the role of the RAmy1A promoter in the temporal andspatial expression of heterologous genes during rice seed germination,histochemical and quantitative GUS assays on transgenic seeds wereperformed. Histochemical analysis of the H4/GUS chimeric gene showedthat GUS activity could be detected in the scutellar epithelium after 2days of germination and that this activity spread into the adjacentaleurone layer by day 4. On day 6, the GUS expression in the aleuronelayer increased to the extent that it covered all portions of the seed.

To quantify the levels of GUS activity revealed by histochemicalanalysis, the GUS activities of germinating seeds derived from twoH4/GUS transgenic lines (T21 and N33) were measured (FIGS. 9A and 9B,respectively). Scutellar GUS activity (open circles) appeared on da 2,peaked on day 4 and decreased thereafter.

In contrast, aleurone GUS activity (closed circles) was first detectedon day 4, peaked on day 6 and decreased sharply by day 8. These resultsclearly show that the RAmy1A1GUS fusion genes are differentiallyregulated in scutellum and aleurone tissues during rice seedgermination.

Similar experiments were performed on seeds from two lines of rice (K43and T62) transformed with the E4/GUS gene fusion (FIGS. 9C and 9D,respectively). Histochemical assays revealed patterns of expressionnearly identical to those observed for the H4/GUS gene. Quantitativeassays of GUS activity in the scutellar and the aleurone layers ofgerminated seeds indicated that the E4/GUS gene is expressed in thescutellum on day 2 and peaks at day 4. GUS activity in the aleuronelayer is first detected at day 4 and peaks on day 6 (FIGS. 9C and 9D).These results show that the -748 to +31 region in the H4/GUS gene andthe -232 to +31 region in the E4/GUS gene, function identically withrespect to the localization of expression and the developmentalregulation during seed germination.

4. Hormonal regulation of the RAmy1A/GUS genes in the aleurone layer oftransgenic rice seeds

To examine effects of exogenously GA and ABA on the expression of theH4/GUS and E4/GUS genes in seeds, deembryonated seeds were cut intothree slices and each slice was treated with GA or a combination of GAand ABA. The histochemical examination of the H4/GUS seed slices treatedwith GA-free buffer showed no GUS activity. However, seed slices treatedwith GA showed GUS activity in the aleurone layer. The observedinduction of the GUS expression by GA was suppressed by addition of ABA.Similar GA induction of GUS in the aleurone layers of pE4/GUStransformed seeds was also observed.

In an attempt to quantify GA induction of the RAmy1A promoter intransgenic seeds, GUS activity was measured for both GA, and GA+ABAtreated seeds (FIGS. 10A-10D). A 10 to 40-fold increase in GUS activitywas observed after GA treatment of H4/GUS (FIGS. 10A and 10B) and E4/GUS(FIGS. 10C and 10D) seeds. When ABA was added along with GA, the GUSactivity was suppressed to a level just above background. The degree ofGA induction and ABA suppression was similar for both H4/GUS and E4/GUSderived seeds.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 7    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 820 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..820    (D) OTHER INFORMATION: /standard.sub.-- name= "HV-18"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GGATCCTAGCTACGGACAGCGCCCCGGTTATGGAGGCCGACAGCCGCGGCGCGCGGCTGC60    GTAGCAGTGCAGCGTGAAGTCATAGATAGACTGTAGAGGGCATGGCGGCAAGTGAAAACA120    CACTTCCGTTTGTTCTGTTGAGTCAGTTGGATCTGCTTTGGCCTGGCGATAACGTCTCCG180    GCCATTGTTTATCACGGCGCCTGCTTATCCCTCCGAAAGTTTGAGCAAAAGGTGCAGCTT240    CTTTCTAGTACAGAAATGACGTCCAGAGTTGCAGCAACCCATTCGGAACTCCTGGTGGAT300    GCCAACGAAATTAAATGGGATAAAACTTAGTGAAGAATCTATATTTTCTTGCAACAACAT360    ACTCCTACCCTCACGAATTGAATGCTCATCGAACGAATGAATATTTGGATATATGTTGAT420    CTCTTCGGACTGAAAAAGTTTGAACTCGCTAGCCACAGCACACTATTCCATGAAAAATGC480    TCGAATGTTCTGTCCTAGAAAAACAGAGGTTGAGGATAACTGACGGTCGTATTGACCGGT540    GCCTTCTTATGGAAGGCGAAGGCTGCCTCCATCTACATCACTTGGGCATTGAATCGCCTT600    TTGAGCTCACCGTACCGGCCGATAACAAACTCCGGCCGACATATCCACTGGCCCAAAGGA660    GCATTCAAGCCGAGCACACGAGAAAGTGATTTGCAAGTTGCACACCGGCAGCAATTCCGG720    CATGCTGCAGCACACTATAAATACTGGCCAGACACACAAGCTGAATGCATCAGTTCTCCA780    TCGTACTCTTCGAGAGCACAGCAAGAGAGAGCTGAAGAAC820    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2389 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..2389    (D) OTHER INFORMATION: /standard.sub.-- name= "RAMY-1A"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CTGCAGGCATGCGAGAGGCACGGGGTTCGATTCCCCGCGTCTCCATCGGCACTGTTTTTT60    AACATCAAACGCTGTTCGATCCACTATCTGTTAATTTCGCAAACACAACTAAATCTTTTT120    TTTTTTTTGCCGGTGCGTGCAGTGTGACGTCCAAGGCATGGCGCATTGGCGCCTCCCCTC180    TTTCCCTTGATCTTTTCATCAGTTCGTTCTTCTTGCAGAAAAGCTGTTCTGTTAAGTCGG240    TTCCGATCTGCTCTTGGGCTCTTGCCAGAAACAACCTGTGTACGCCAGACTTATCAAGCC300    AACCATCCTGATGAGCCTCTGCTTATACAAGCCTTTGACTCCAAAAAGGACGAGGAGGCT360    TGCAGCCGCACGGAAATAAGCCGACCGATCCTTTATTGCTCTATCTTTTTCCCTTGGAAT420    AAAAAACAGCCCAATTAAAATCTGGGATGAAACTATGGCTAGCTGTTCGCGGTGTCAGTT480    CTCGGGACGCTACCGTTGTTTTGTTTGAACCGGAATGTTCAGGGCGGTTCACACCATAGA540    CTTGGAGCCAAGTGGTTCCATCCACAAAATTTTCTCATCTTGAATATTCTGTTATCTGCC600    TCGACAGACGCACCATATCCTGTGTTCAGGAATGAATGTGCTACAGCCAACGTGCTGCAT660    GAAATTTGCTGAAATCGTGCTAAAATGTGCATGGCAACAGGAACCTGATGCCCTGGTCCT720    GTGGAACTGCCACGGGAAAGTATTTTTTATAGCTAGGTGCAATCGTATCTAGGTGTATAC780    ATGTCACCTACATAGCTACTCCCCTTTATCTTAAAATATAATAATTTTTAACTCTCAGTA840    TTTGTCCTAAAATATAACAAATTCTCCATCAACATTATCTTCCCAACCAATCACAACCCT900    TCATCATTAATTTTTTCCCCCTACCTCCACTACTCATCTAATCACAACCCTCCAACACTC960    ACTTCTATCTACTTTCTTAATAACTGTCTTCAACCCTAAAACTTCTTATATTTTAGGACG1020    GAGGGAGTATCTAAATATTTCATAAAAAAAATGTTAAGATAGATAAAGAAGATATAAACC1080    CACTATGCAAACATGCACATCAAAATTTAATTTACAGTAAAGAAACAGAAATAACATATT1140    CTATTTGTGCTGGAGATGTACTGTTCACAATATTGTTTTTTTATTTTTTATTTATCTGAT1200    TATATATCTGTTTCAGCCTTGCATGGTTGTGTATGTTTGTGTATAGACTTATGCCATTGT1260    GATTGATGCTACCAATTATTTTCAGACTATTTTTTTATAGAGGAATTTTATAGTTCTTGA1320    GAAAATACCTTGAAGTATCTAAATTTTACACTAAAATTGTTGGTACCTTGAGGTACAAAG1380    TACCTAGAGGTACCAAATTTTACTAGAAAATTGTGGCACCTTTAGGTACCTTCTCAAAAA1440    TAGTACAATTATGGGCCGTTTTGGATTTAGTGCCAAAACGTGCTCTACAAATATTTTGAT1500    AGTTTGAACAGTGCATAAGACGGGTTTGGTTTGAAGCCAAATCATTGGCATTGCCAATGT1560    CCAATTTGATATTTTCTATATTATGCTAAAAGCTTGGTTCTAAATTGGCCTCCAACCAAA1620    TACAACTCTACTCTACCAAAAAATTTGTAGTGCCAAAACTTGCCTAGGTTTTGTCACTAC1680    CAACATTTTGGTAAGTATTAAACCAAACAAGCCCTACATTTTTTTATGTACATTTAAGTT1740    GTATGTAAATGATGGGTGCGGTTGCACCTAGGTGAAAAAAAATACATATTCGCCACAACT1800    CGCAACATGTACCAATTCAGCAGCAAGTGTAAGAGAGAAGATTTCTCTCGTTTTACACGC1860    GCACGTTCAATTCCTGAACTACTAAACGGTATGATTTTTTGCAAAAATTTTCTATAGGAA1920    AGTTACTTAAAAATTATATTAATCTATTTTTAAAATTTAAAATAGTTAATACTCAATTAA1980    TTATACGTTAATGGCTCAGCTCGTTTTGCGTACATTCTCAATCGATTCTTTTCCTCTGCT2040    CTCAAATGCTCTGTGTGCGATCAGGTATTCATGTTCAGCTCGCACAAGCACAAGCAAGAC2100    AGATGGAATTCCTACTGACCTGCGCCTTTTGAGTCGCTCCAACTCTCAAAGTCTCAAGGC2160    CATTAAATTGCCTATGGGCTCACCAGCCAATAACAAACTCCGGCTGTTATCCATCCAATC2220    CAGTGTCCCAAAGCAACATTCAAGCCCAGCCAGGCCTCCAAAAGTTGCAAGTTGAGCATG2280    GCAAAATCCCCGGCAATTCTCGACTATAAATACCTGACCAGACACACCCAGGAGCTTCAT2340    CAATCATCCATCTCCGAAGTGTGTCTGCAGCATGCAGGTGCTGAACACC2389    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..31    (D) OTHER INFORMATION: /standard.sub.-- name= "31 bp RAmy3D"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GAGACCGGGCCCCGACGCGGCCGACGCGGCG31    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..31    (D) OTHER INFORMATION: /standard.sub.-- name= "31 bp RAmy3E"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GAGAGCTCGCGCCGCCTCGATCGGCGCGGCG31    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /standard.sub.-- name= "11 bp RAmy3D"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    TTCCGGCTTGC11    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /standard.sub.-- name= "11 bp RAmy3E"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    TTGCGGCTTGC11    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..12    (D) OTHER INFORMATION: /standard.sub.-- name= "Taka-amylase"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    CGGCCCGTCGGC12    __________________________________________________________________________

It is claimed:
 1. A transgenic monocot plant seed having an endospermsurrounded by an aleurone or scutellar epithelium layer, said seed beingstably transformed with a chimeric gene having (i) a transcriptionalregulatory region inducible during seed germination, by adding orremoving a small molecule, (ii) a heterologous first DNA sequenceencoding a protein, and (iii) a second DNA sequence encoding a signalpolypeptide, where said second DNA sequence is operably linked to saidtranscriptional regulatory region and said first DNA sequence, and wheresaid signal polypeptide is in translation-frame with said protein and iseffective to facilitate secretion of said protein across said aleuroneor scutellar epithelium layer into the endosperm,wherein adding orremoving said small molecule to said seeds, during seed malting, iseffective to upregulate said transcriptional regulatory region, therebyto induce expression of said protein and secretion of said protein fromthe aleurone or scutellar cells into the seed endosperm.
 2. The seed ofclaim 1, wherein the seed is from a cereal plant selected from the groupconsisting of wheat, rice, oats, rye, corn, sorghum, millet and barley.3. The seed of claim 2, wherein the cereal seed is barley.
 4. The seedof claim 2, wherein the cereal seed is rice.
 5. The seed of claim 1,wherein the regulatory region is from an α-amylase gene selected fromthe group consisting of RAmy1A (SEQ ID NO:2), RAmy3B, RAmy3C, RAmy3D,HV18 (SEQ ID NO:1) and RAmy3E, and RAmy2A.
 6. The seed of claim 5, wheresaid promoter is from the RAmy3D gene or a homolog thereof.
 7. The seedof claim 5, where said promoter is from the α-amylase RAmy1A or HV18gene or a homolog thereof.
 8. The seed of claim 1, wherein the proteinis a growth factor.
 9. The seed of claim 1, wherein the protein is avaccine.
 10. A plant seed composition produced by (i) malting the seedsof claim 1 under conditions in which said small molecule is added orremoved to induce heterologous protein expression, and (ii) forming amash containing endosperm tissue from the seed.
 11. The composition ofclaim 10, wherein the heterologous protein is a growth hormone.
 12. Thecomposition of claim 10, wherein the heterologous protein is a vaccine.